CN106770806A - It is a kind of to determine aflatoxin B1, the method for B2 contents in Chinese patent drug simultaneously - Google Patents
It is a kind of to determine aflatoxin B1, the method for B2 contents in Chinese patent drug simultaneously Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides aflatoxins B in a kind of Chinese patent drug of measure simultaneously1, B2The method of content, the adsorbent that the present invention is extracted using the aminopropyltriethoxywerene werene of tetraethyl orthosilicate 3 modification ferroferric oxide magnetic nanoparticle as Magnetic solid phases, is used in AFB first1, B2Enrichment in, the features such as with easy to operate, low cost, the degree of accuracy high；The highly sensitive magnetic nanoparticle stationary phases for HPLC that the present invention is set up, to AFB in the Chinese patent drug with complex material system1, B2Assay research is carried out simultaneously, has the advantages that low cost, selectivity are high, preferably to ensure that Chinese native medicine security provides technical support.
Description
(1) technical field
Aflatoxins B is determined simultaneously the present invention relates to a kind of1, B2The method of content, more particularly to it is a kind of determine simultaneously in into
Aflatoxins B in medicine1, B2The method of content.
(2) background technology
Aflatoxins is the fungal secondary metabolite that one group of basic structure is two furan nucleus blending legumins, and common Huang is bent
Mycin has aflatoxins B1(AFB1)、B2(AFB2)、G1(AFG1)、G2(AFG2), with poison such as serious carcinogenic, teratogenesis, mutagenesis
Side effect.Because of its hypertoxic and strong carcinogenicity, aflatoxins turns into domestic and international government and consumer's supervision and focal point.Chinese medicine
Aflatoxins pollution is increasingly paid close attention to by international community in recent years.This is that, because Chinese medicine species is various, plantation area is wide
General, most medicinal materials are during production, processing, storage, transport, it is easy to go mouldy and polluted by aflatoxins.And in
Patent medicine not yet has standard to specify the limitation of its aflatoxins as the Major Clinical type of service of Chinese medicine.So being directed to
Chinese patent drug sets up highly sensitive aflatoxins assay method urgent demand.
Magnetic solid phases extraction (MSPE) are a kind of sample-pretreating methods with magnetic material as adsorbent.Magnetic Nano material
Material can be used as the adsorbent of MSPE, it is advantageous that：The specific surface area of nano-particle is big, diffusion length is short, only needs to use
A small amount of adsorbent and shorter equilibration time can be achieved with extract and separate, therefore be imitated with extracting power higher and extraction
Rate.MSPE technologies have been applied in contaminant trace species extract and separate field, are concentrated mainly on to being metabolized product in biological sample
In the enrichment of thing or metal ion, but use the application study of sample pre-treatments in Chinese medicine less.What this seminar had applied
The functional magnetic nano material 2- amino-5- sulfydryl-1,3,4- thiadiazoles of patent of invention 201610528482.7-(3- sulfydryls third
Base) trimethoxy silane-magnetic nanoparticle be used for Chinese patent drug Shengmai Yin (Radix Codonopsis side) in AFB1Enrichment at
Reason, has obtained more satisfied result.On this basis, this seminar further explores different functional magnetic nanometer materials
Material, and it is used in different Chinese patent drug sample aflatoxins B1And B2While determine, it is strong to pass through to find selectivity
Functional magnetic nano particle, the condition to Magnetic solid phases extraction optimizes, set up the extraction of highly sensitive Magnetic solid phases-
The method of high performance liquid chromatography-fluorescence is used for aflatoxins B in Chinese patent drug1, B2Assay, be Chinese patent drug aspergillus flavus
The control of element and the formulation of limit standard provide method, are exploitation preferably to ensure that Chinese native medicine security provides technical support
A kind of low cost, the aflatoxins sample pretreatment material of high selectivity provide foundation.
(3) content of the invention
Magnetic nanoparticle is used for aflatoxins B in measure Chinese patent drug simultaneously it is an object of the present invention to provide one kind1, B2Contain
The method of amount, the characteristics of the inventive method has quick, sensitive, inexpensive.
The technical solution adopted by the present invention is：
It is a kind of to determine aflatoxins B in Chinese patent drug simultaneously1, B2The method of content, described method is：
(1) need testing solution is prepared
Take during Chinese patent drug to be measured adds pure water, ultrasonic disperse is uniform, regulation pH to 2~5 (preferably 3, with 20wt%~
37wt% hydrochloric acid carries out pH regulations), magnetic nanoparticle is subsequently adding, stirring and adsorbing obtains mixed liquor；Gained mixed liquor is through outer
Plus magnetic fields layering, incline except supernatant liquor, dichloromethane and acetone volume ratio 2 are added in lower sediment：1 mixing is molten
Agent, stir desorption, after through externally-applied magnetic field act on be layered, collect supernatant liquor, after volatilizing naturally at room temperature, surplus materials use
Absolute methanol redissolves, filter membrane (0.22 μm of aperture), collects filtrate and obtains final product need testing solution (concentration range is：0.3~3.4ng
Surplus materials/mL absolute methanols)；
The volumetric usage of the pure water is calculated as 3~7mL/g, preferably 5mL/g with the quality of Chinese patent drug to be measured；
Described Chinese patent drug to be measured is solid pharmaceutical preparation or liquid preparation；When Chinese patent drug to be measured is solid pharmaceutical preparation, the magnetic
Property nano particle and Chinese patent drug to be measured mass ratio be 0.01~0.02：1, preferably 0.015：1；When Chinese patent drug to be measured is liquid system
During agent, the quality consumption of the magnetic nanoparticle is calculated as 10~20mg/mL, preferably 15mg/mL with the volume of Chinese patent drug to be measured；
The time of the stirring and adsorbing is 5~15min, preferably 10min；
The dichloromethane and acetone volume ratio 2：The volumetric usage of 1 mixed solvent is calculated as with the quality of Chinese patent drug to be measured
0.3~1.8mL/g, preferably 1.2mL/g；
The time of the stirring desorption is 2~10min, preferably 10min；
Described externally-applied magnetic field refers to using magnet as externally-applied magnetic field source；
(2) need testing solution detection
The need testing solution for taking step (1) preparation carries out high performance liquid chromatography detection, obtains the efficient liquid of need testing solution
Phase chromatogram；The testing conditions of the high performance liquid chromatography are：Carbon octadecylsilane chemically bonded silica with particle diameter≤5 μm is as color
Spectrum column packing；With acetonitrile, methyl alcohol, the aqueous formic acid of volumetric concentration 0.1% mixed liquor as mobile phase, flow velocity：0.8~
1.2mL/min；Column temperature：20~30 DEG C；Fluorescence detector；Number of theoretical plate presses aflatoxins B1It is (logical that peak calculating should be not less than 3000
Often 3000~10000)；
In described mobile phase, acetonitrile, methyl alcohol, the percentage by volume difference of the aqueous formic acid three of volumetric concentration 0.1%
It is 10%~25%, 10%~25%, 50%~80%, the percentage by volume of preferably three is respectively 20%, 20%, 60%；
Described chromatographic column is C18Post, 4.6mm × 250mm, preferably described C18Filler carbon octadecylsilane key in post
The particle diameter for closing silica gel is 5 μm or 3.5 μm；
It is preferred that the column temperature：25℃；
The fluorescence detector excitation wavelength：360nm, launch wavelength：440nm；
(3) standard curve is set up
Weigh aflatoxins B1, B2Standard items, hybrid standard product solution is configured to methyl alcohol, takes different amounts of hybrid standard
Product solution, is detected respectively under the conditions of with step (2) identical high performance liquid chromatography detection, obtains hybrid standard product solution
High-efficient liquid phase chromatogram；With hybrid standard product solution Plays product aflatoxins B1, B2Respective sample introduction quality be abscissa,
The high-efficient liquid phase chromatogram Plays product aflatoxins B of hybrid standard product solution1, B2The peak area of respective absworption peak is vertical seat
Mark, draws standard curve, carries out linear regression, obtains aflatoxins B1, B2Respective standard curve and equation of linear regression；
In the hybrid standard product solution, standard items aflatoxins B1Concentration be 0.3~10ng/mL, standard items are yellow bent
Mycin B2Concentration be 0.03~5ng/mL；
(4) testing sample result is determined
By aflatoxins B in the high-efficient liquid phase chromatogram of step (2) need testing solution1, B2The peak area number at respective absorption peak
Value is substituted into the equation of linear regression that step (3) is set up, and is calculated aflatoxins B in need testing solution1, B2Content, and then
Extrapolate aflatoxins B in Chinese patent drug to be measured1, B2Content.
In the present invention, described magnetic nanoparticle is：Tetraethyl orthosilicate-APTES modification four
Fe 3 O magnetic nanoparticle, is denoted as：APTES-TEOS-MNPs, wherein TEOS represent tetraethyl orthosilicate, and APTES represents 3-
Aminopropyltriethoxywerene werene, MNPs represents ferroferric oxide magnetic nanoparticle.
Described APTES-TEOS-MNPs magnetic nanoparticles are prepared as follows obtaining：
A () is by FeCl3·6H2O、FeCl2·4H2O mixes with deionized water, and under nitrogen protection, 85 DEG C of heating water baths are simultaneously
Stirring 30min, is then injected into ammoniacal liquor (25wt%~28wt%), then continues to react 30min in 85 DEG C, is centrifuged afterwards, and it is heavy to take
Shallow lake is washed with deionized water, 0.02mol/L sodium-chloride water solutions successively, and 36h is dried at 40~60 DEG C, obtains ferroso-ferric oxide magnetic
Property nano particle；
The FeCl3·6H2O and FeCl2·4H2The mass ratio of O is 2~3：1；
The volumetric usage of the deionized water is with FeCl3·6H2O and FeCl2·4H2The gross mass of O is calculated as 12~13mL/
g；
The volumetric usage of the ammoniacal liquor is with FeCl3·6H2O and FeCl2·4H2The gross mass of O is calculated as 1.4~1.8mL/g；
B step (a) gained ferroferric oxide magnetic nanoparticle is dissolved in methyl alcohol and pure water volume ratio 3~4 by ()：1
In mixed solution, ultrasonic power be 600W under conditions of ultrasound 10min, be subsequently adding ammoniacal liquor (25wt%~28wt%),
Tetraethyl orthosilicate (TEOS), is stirred at room temperature 10h, after be layered through externally-applied magnetic field, incline except supernatant, collect precipitation and be placed in 40~60
24h is dried in DEG C vacuum drying chamber, TEOS-MNPs magnetic nanoparticles are obtained after grinding；
The methyl alcohol is calculated as 200 with the volumetric usage of pure water mixed solution with the quality of ferroferric oxide magnetic nanoparticle
~300mL/g；
The volumetric usage of the ammoniacal liquor is calculated as 20~30mL/g with the quality of ferroferric oxide magnetic nanoparticle；
The volumetric usage of the TEOS is calculated as 0.5~1.0mL/g with the quality of ferroferric oxide magnetic nanoparticle；
(c) by step (b) gained TEOS-MNPs magnetic nanoparticles and APTES (APTES),
Toluene mixes, and is warming up to 110 DEG C of backflow 10h, after be layered through externally-applied magnetic field, incline except supernatant, collect precipitation and be placed in 45~60
24h is dried in DEG C vacuum drying chamber, described APTES-TEOS-MNPs magnetic nanoparticles are obtained final product after grinding；
The volumetric usage of the APTES is calculated as 10~15mL/g with the quality of TEOS-MNPs magnetic nanoparticles；
The volumetric usage of the toluene is calculated as 100~150mL/g with the quality of TEOS-MNPs magnetic nanoparticles.
Heretofore described room temperature is 20~30 DEG C.
Compared with prior art, the present invention has advantages below and beneficial effect：
(1) highly sensitive magnetic nanoparticle-stationary phases for HPLC is set up to complex material system
AFB in Chinese patent drug1, B2Assay research is carried out simultaneously, has the advantages that low cost, selectivity are high；
(2) functional magnetic nano particle is mainly concentrated use in metabolite or gold in biological sample and food at present
Belong in the enrichment of ion, the present invention explores the adsorbent that a kind of APTES-TEOS-MNPs is extracted as Magnetic solid phases, first will
It is used in AFB1, B2Enrichment in, have the advantages that easy to operate, low cost, the degree of accuracy are high.
(4) illustrate
Fig. 1：Different factors are to aflatoxins B1, B2The influence of concentration effect, (a) sample solution pH, (b) magnetic Nano
Grain addition, (c) enrichment time, (d) eluant, eluent species【1 to 4 are respectively CH2Cl2:Me2CO(1:1, v/v), CH2Cl2:Me2CO
(2:1, v/v), CH2Cl2:MeCN(1:1, v/v), CH2Cl2:MeOH(1:1, v/v)】, (e) effluent volume, (f) elute when
Between；
Fig. 2：Aflatoxins B1, B2The liquid chromatogram of reference substance；
Fig. 3：The liquid chromatogram of the need testing solution of embodiment 4；
Fig. 4：The liquid chromatogram of Fupoganmao particle under different flow phase systems, A is the formic acid water of acetonitrile-methanol -0.1%
=10:10:80, B is formic acid water=15 of acetonitrile-methanol -0.1%:15:70, C is formic acid water=20 of acetonitrile-methanol -0.1%:
20:60；
Fig. 5：The liquid chromatogram of Fupoganmao particle under the conditions of different column temperatures, D is 20 DEG C of column temperature, and E is 25 DEG C of column temperature, F
For 30 DEG C of column temperature；
Fig. 6：The liquid chromatogram of different Chinese patent drug samples, 1 to 12 are respectively：Shengmai Yin (Radix Codonopsis side), ageratum glue
Capsule, Fupoganmao particle, tropaeolum oral liquid, antiageing tablet, logical a surname's oral liquid, join its hypoglycemic granule, protect and oral liquid, jade screen
Oral liquid, wheat god powder for improving appetite, oral stomachic tonic liquor, wushicha granules.
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
Fupoganmao particle used is purchased from certain pharmaceutical Co. Ltd in embodiment of the present invention 2-4.
Externally-applied magnetic field described in the embodiment of the present invention presses close to round-bottomed flask as outer using the magnet piece of 150*100*25mm
Plus magnetic field sources.
The preparation of the magnetic nanoparticle of embodiment 1
(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, and 85 DEG C of heating water baths and 30min is stirred under conditions of nitrogen protection, is slowly injected into matter
The ammoniacal liquor 23mL of concentration 25% is measured, solution colour is changed into black from orange.After adding ammoniacal liquor to terminate, continue to heat, 85 DEG C of reactions
30min, centrifugation, resulting precipitation is washed with deionized 2 times, then is washed 1 time with 0.02mol/L sodium-chloride water solutions, obtains
To particle be deposited in 36h dried at 45 DEG C, that is, obtain ferroferric oxide magnetic nanoparticle (MNPs) 1.5g, it is standby.
(2) ferroferric oxide magnetic nanoparticle 1.0g is taken, it is 4 to be dissolved in methyl alcohol with pure water volume ratio:1 250mL is mixed
Close in solution, the ultrasound 10min under conditions of ultrasonic power is 600W adds the ammoniacal liquor of 30mL 25%, is slowly added to 800 μ
L TEOS, are stirred at room temperature 10h, and its fast hierarchical is made with externally-applied magnetic field, remove supernatant, and the particle for obtaining is deposited in 60 DEG C
24h is dried in vacuum drying chamber, dry particle grinds to form fine powder, that is, obtain TEOS-MNPs magnetic nanoparticles.
(3) the TEOS-MNPs 0.5g of above-mentioned synthesis are taken, are added in 100mL round-bottomed flasks, and add 6mL APTES,
110 DEG C of backflow 10h in 60mL toluene solutions.Make its fast hierarchical with externally-applied magnetic field, remove supernatant, the particle precipitation for obtaining
24h is dried in 45 DEG C of vacuum drying chambers, dry particle grinds to form fine powder, that is, obtain the ethoxy of tetraethyl orthosilicate 3- aminopropyls three
Base silane modified by magnetic nanoparticles thing (APTES-TEOS-MNPs) 0.6g, uses for sample pre-treatments.
The optimization of the chromatographic condition of embodiment 2
1st, sample pre-treatments：Precision weighs Fupoganmao particle 10g, is dissolved in 50mL pure water, is 600W in ultrasonic power
Under conditions of ultrasound 3min, adjust pH=3, the APTES-TEOS-MNPs magnetic Nanos for adding the method for 150mg embodiments 1 to prepare
Particle, stirs 10min at room temperature, and magnetic nanoparticle is separated with supernatant with externally-applied magnetic field, pours out supernatant, and precipitation is added
12mL CH2Cl2:Me2CO(2:1, v/v) desorption 10min, is stirred, in magnetic fields lower leaf, supernatant liquor is collected, it is natural
Redissolved with 300 μ L absolute methanols after volatilizing, cross 0.22 μm of filter membrane, take filtrate, sample solution is obtained.
2nd, mobile phase selection
Aflatoxins B is carried out to sample solution using stationary phases for HPLC1, B2Assay.
(1) instrument and reagent：
Ultra Performance Liquid Chromatography instrument (Agilent Technologies 1290infinity, including quaternary gradient pump
(G4204A, 1290Quat Pump), on-line degassing machine (G1316C, 1290TCC), automatic sampler (G4226A, Sample),
Column oven (G1330B, 1290Thermostat), fluorescence detector (G1321B, 1260FLD), Data Processing in Chromatography Workstation)；Chromatogram
Post：C18 posts (Agilent Eclipse SB-C18,4.6mm × 250mm, 5 μm)；Ten a ten thousandth electronic balance (METTLER
XS205)；Fourier trasfonn infrared microscope spectrometer (Nicolet 6700)；ESEM (Hitachi S-4160)；Ao Haosi
AR224w (a ten thousandth electronic balance)；Formic acid (chromatographically pure, TEDIA companies of the U.S.)；(chromatographically pure, U.S. TEDIA is public for acetonitrile
Department)；FeCl3·6H2O (Chemical Reagent Co., Ltd., Sinopharm Group)；FeCl2·4H2O (Shanghai fuzz Chemical Co., Ltd.)；Just
Silester [ethyl silicate, TEOS] (Aladdin biochemical technology limited company, purity >=95%)；3- aminopropans
Ethyl triethoxy silicane alkane [(3-aminopropyl) triethoxysilane, APTES] (limited public affairs of Aladdin biochemical technology share
Department, purity >=98.0%)；Dichloromethane, acetone, ammoniacal liquor are AG (Hangzhou Long March chemical reagent Co., Ltd)；Huang Qu
Mould toxin B1、B2(Sigma Chemical companies), detects that purity is more than 98% through efficient liquid phase；Water is pure water；
Afla-P Aspergillus flavus toxin immuno-affinity columns (Vicam companies of the U.S.).
(2) selection of chromatographic condition
Chromatographic column：Agilent SB-C18Post (4.6mm × 250mm, 5 μm)；
Mobile phase is respectively：The aqueous formic acid (10 of acetonitrile-methanol-volumetric concentration 0.1%:10:80, v/v/v), acetonitrile-
The aqueous formic acid (15 of methyl alcohol-volumetric concentration 0.1%:15:70, v/v/v), the formic acid of acetonitrile-methanol-volumetric concentration 0.1% is water-soluble
Liquid (20:20:60, v/v/v) isocratic elution, is carried out respectively；
Flow velocity：1.0mL/min；
Column temperature：25℃；
Detector：Fluorescence detector；
Detection wavelength：Excitation wavelength：360nm, launch wavelength：440nm；
Sample size：20μL.
Result shows that (see Fig. 4) uses the aqueous formic acid (20 of acetonitrile-methanol-volumetric concentration 0.1%:20:60, v/v/v) it is
Mobile phase isocratic elution effect is more excellent, and each chromatographic peak separating degree is good, and peak shape is sharp.
3rd, the influence of column temperature
With reference to the selection of mobile phase, column temperature is respectively：20℃、25℃、30℃；Mobile phase is：Acetonitrile-methanol-volume is dense
Spend 0.1% aqueous formic acid (20:20:60, v/v/v).
Result shows (see Fig. 5), and the separation feelings of chromatographic peak under different column temperatures (20 DEG C, 25 DEG C, 30 DEG C) have been investigated in experiment
Condition is similar to, and separation influence of the temperature on chromatographic peak is little.
The optimization of the magnetic nanoparticle extraction conditions of embodiment 3
1st, sample solution pH：Precision weighs 5 parts of each 10g of Fupoganmao particle, is dissolved in 50mL pure water, is in ultrasonic power
Ultrasound 3min, 1.0,3.0,5.0,7.0,9.0 are tuned into by its pH respectively under conditions of 600W, to being uniformly added into sample solution
Magnetic nanoparticle prepared by the method for 150mg embodiments 1, stirs 10min, with externally-applied magnetic field by magnetic nanoparticle and supernatant
Separate, pour out supernatant liquor, lower sediment 9mL CH2Cl2:Me2CO(2:1, v/v) desorption 10min, is stirred, is made in magnetic field
With lower leaf, supernatant liquor is collected, redissolved with 300 μ L absolute methanols after volatilizing naturally, cross 0.22 μm of filter membrane, take filtrate, be obtained
Sample solution.
High performance liquid chromatograph detection (column temperature is carried out using the method for embodiment 2：25 DEG C, the formic acid water of acetonitrile-methanol -0.1%
(20:20:60, v/v/v) it is mobile phase), as a result show the solution condition aflatoxins B of (see a in Fig. 1) pH=31, B2Absorption
Effect is more excellent.
2nd, magnetic nanoparticle consumption：Precision weighs 5 parts of each 10g of Fupoganmao particle, 50mL pure water is dissolved in, in ultrasonic wave
Power is ultrasound 3min under conditions of 600W, regulation pH=3.0, to being separately added into 50mg, 100mg, 150mg in sample solution,
The method of 200mg, 250mg embodiment 1 prepare magnetic nanoparticle, stir 10min, with externally-applied magnetic field by magnetic nanoparticle with
Supernatant is separated, and pours out supernatant liquor, lower sediment 9mL CH2Cl2:Me2CO(2:1, v/v) desorption 10min, is stirred,
Magnetic fields lower leaf, collects supernatant liquor, is redissolved with 300 μ L absolute methanols after volatilizing naturally, crosses 0.22 μm of filter membrane, takes filter
Liquid, is obtained sample solution.
High performance liquid chromatograph detection (column temperature is carried out using the method for embodiment 2：25 DEG C, the formic acid water of acetonitrile-methanol -0.1%
(20:20:60, v/v/v) it is mobile phase), as a result show that (see b in Fig. 1) is 10g, amount of particles 150mg when test sample consumption is treated
When, AFB can be made1, B2Absorption is complete.
3rd, magnetic nanoparticle adsorption time：Precision weighs 5 parts of each 10g of Fupoganmao particle, is dissolved in 50mL pure water, super
Acoustic power is ultrasound 3min under conditions of 600W, adjusts pH=3.0, to the method system of addition 150mg embodiments 1 in sample solution
Standby magnetic nanoparticle, stirs 5min respectively, and 8min, 10min, 12min, 15min externally-applied magnetic field are by magnetic nanoparticle
Separated with supernatant, pour out supernatant liquor, lower sediment 9mL CH2Cl2:Me2CO(2:1, v/v) desorption 10min, is stirred,
In magnetic fields lower leaf, supernatant liquor is collected, redissolved with 300 μ L absolute methanols after volatilizing naturally, cross 0.22 μm of filter membrane, take filter
Liquid, is obtained sample solution.
High performance liquid chromatograph detection (column temperature is carried out using the method for embodiment 2：25 DEG C, the formic acid water of acetonitrile-methanol -0.1%
(20:20:60, v/v/v) it is mobile phase), as a result show (see c in Fig. 1) adsorption time for 10min is more excellent.
4th, different eluant, eluents：Precision weighs Fupoganmao particle 10g, is dissolved in 50mL pure water, is 600W in ultrasonic power
Under conditions of ultrasound 3min, pH=3.0 is adjusted, to the magnetic Nano for adding the method for 150mg embodiments 1 to prepare in sample solution
Grain, stirring 10min externally-applied magnetic fields separate magnetic nanoparticle with supernatant, pour out supernatant liquor, and lower sediment is used respectively
12mL CH2Cl2:Me2CO(2:1, v/v), CH2Cl2:Me2CO(1:1, v/v), CH2Cl2:MeCN(1:1, v/v), CH2Cl2:
MeOH(1:1, v/v) desorption 10min is stirred, in magnetic fields lower leaf, supernatant liquor is collected, with 300 μ L after volatilizing naturally
Absolute methanol redissolves, and crosses 0.22 μm of filter membrane, takes filtrate, and sample solution is obtained.
High performance liquid chromatograph detection (column temperature is carried out using the method for embodiment 2：25 DEG C, the formic acid water of acetonitrile-methanol -0.1%
(20:20:60, v/v/v) it is mobile phase), as a result show that (see d in Fig. 1) eluant, eluent is CH2Cl2:Me2CO(2:1, v/v) washed when
De- effect is more excellent.
5th, effluent volume：Precision weighs 5 parts of each 10g of Fupoganmao particle, is dissolved in 50mL pure water, is in ultrasonic power
Ultrasound 3min under conditions of 600W, adjusts pH=3.0, is received to the magnetic for adding the method for 150mg embodiments 1 to prepare in sample solution
Rice grain, stirring 10min externally-applied magnetic fields separate magnetic nanoparticle with supernatant, pour out supernatant liquor, lower sediment point
Yong not 3mL, 6mL, 9mL, 12mL, 18mL CH2Cl2:Me2CO(2:1, v/v) desorption 10min, is stirred, is divided under magnetic fields
Layer, collects supernatant liquor, is redissolved with 300 μ L absolute methanols after volatilizing naturally, crosses 0.22 μm of filter membrane, takes filtrate, sample is obtained molten
Liquid.
High performance liquid chromatograph detection (column temperature is carried out using the method for embodiment 2：25 DEG C, the formic acid water of acetonitrile-methanol -0.1%
(20:20:60, v/v/v) it is mobile phase), as a result show (see e in Fig. 1) effluent volume for 12mL is more excellent.
6th, the desorption time：Precision weighs 5 parts of each 10g of Fupoganmao particle, is dissolved in 50mL pure water, is in ultrasonic power
Ultrasound 3min under conditions of 600W, adjusts pH=3.0, is received to the magnetic for adding the method for 150mg embodiments 1 to prepare in sample solution
Rice grain, stirring 10min externally-applied magnetic fields separate magnetic nanoparticle with supernatant, pour out supernatant liquor, lower sediment point
12mL CH are not used2Cl2:Me2CO(2:1, v/v) desorption 2min, 4min, 6min, 8min, 10min, are stirred respectively, in magnetic field
Effect lower leaf, collects supernatant liquor, is redissolved with 300 μ L absolute methanols after volatilizing naturally, crosses 0.22 μm of filter membrane, takes filtrate, makes
Obtain sample solution.
High performance liquid chromatograph detection (column temperature is carried out using the method for embodiment 2：25 DEG C, the formic acid water of acetonitrile-methanol -0.1%
(20:20:60, v/v/v) it is mobile phase), as a result show (see f in Fig. 1) the desorption time for 10min is more excellent.
Aflatoxins B in the Chinese patent drug of embodiment 41, B2Assay
(1) chromatographic condition and system suitability test
Instrument is with reagent with embodiment 2.
(2) selection of chromatographic condition
By to the chromatographic conditions such as instrument, mobile phase, Detection wavelength, column temperature optimize investigation on the basis of, obtain
Can be suitably used for aflatoxins B1, B2The chromatographic condition of assay：
Chromatographic column：Agilent Eclipse SB-C18Post (4.6mm × 250mm, 5 μm)；
Mobile phase：By the formic acid water of acetonitrile-methanol -0.1% (20:20:60, v/v/v) isocratic elution is carried out；
Flow velocity：1.0mL/min；
Column temperature：25℃；
Detector：Fluorescence detector；
Detection wavelength：Excitation wavelength：360nm, launch wavelength：440nm；
Sample size：20μL.
(3) preparation of standard solution：
Precision weighs aflatoxins B1, B2Appropriate standard items, concentration respectively B is configured to methyl alcohol1：10ng/mL, B2：
5ng/mL standard solutions, high-efficient liquid phase chromatogram is shown in Fig. 2.
(4) preparation of need testing solution：
Precision weighs Fupoganmao particle 10g, is dissolved in 50mL pure water, the ultrasound under conditions of ultrasonic power is 600W
3min, adjusts pH=3.0, to the magnetic nanoparticle for adding the method for 150mg embodiments 1 to prepare in sample solution, stirs 10min
Magnetic nanoparticle is separated with supernatant with externally-applied magnetic field, pours out supernatant liquor, lower sediment 12mL CH2Cl2:Me2CO
(2:1, v/v) desorption 10min, is stirred, in magnetic fields lower leaf, supernatant liquor is collected, it is anhydrous with 300 μ L after volatilizing naturally
Methyl alcohol redissolves, and crosses 0.22 μm of filter membrane, and need testing solution is obtained, and enters chromatogram such as Fig. 3 of liquid phase detection.
(5) drafting of standard curve
It is accurate respectively to draw the μ L of standard solution 1,2 μ L, 5 μ L, 10 μ L, 20 μ L injection high performance liquid chromatographs, measure peak
Area.With the quality (X, ng) of aflatoxin contained in standard solution for abscissa, peak area (Y, mAU) is vertical seat
Mark, draws standard curve, carries out linear regression, obtains regression equation and is shown in Table 1.
The aflatoxin standard curve of table 1
(6) precision test
Under above-mentioned chromatographic condition, precision draws standard solution interior repetition sample introduction 6 times on the same day.Result calculates aspergillus flavus
Plain B1, B2The RSD of peak area is respectively 1.2%, 2.3%.Under above-mentioned chromatographic condition, precision draws standard solution, continuous 3
Its sample introduction 1 time daily.Result calculates aflatoxins B1, B2The RSD of peak area is respectively 1.2%, 1.9%.Result above shows instrument
Device precision is good.
(7) stability test
Precision draws same need testing solution, and peak area is determined respectively at 2,4,8,16,24h, as a result small in 24 for determining
When it is interior, calculate aflatoxins B1, B2Peak area RSD values are respectively 1.1%, 1.8%.Show need testing solution in 24h internal stabilities
Well.
(8) replica test
Precision weighs 6 parts of same Fupoganmao particle sample, and prepared by the method according to step (4) need testing solution, determine knot
Fruit calculates aflatoxins B1, B2The RSD of peak area is respectively 1.4%, 2.7%.Show that the repeatability of sample is good.
(9) recovery test
Using sample-adding recovery experiment, precision takes the B of 10ng/mL1Standard solution 6 parts of each 0.125ml, the B of 5ng/ml2Mark
6 parts of each 0.06mL of quasi- product solution, mixing, 50mL is settled to pure water.6 parts of each 5g of Fupoganmao particle are poured into respectively, by step
(4) prepared by the method for need testing solution, determines, and calculates the rate of recovery, average recovery B1, B2Respectively 98.6 ± 2.5%, 95.9
± 2.1%.Show that the method accuracy is good.
(10) sample size is determined
The Chinese patent drug samples different to 12 kinds carry out aflatoxins B1, B2Determine, the results are shown in Table 2.
The chromatogram of 12 kinds of Chinese patent drug samples is shown in Fig. 6.
The different dosage forms Chinese patent drug AFB of table 21, B2Assay result (ng/g or ng/mL)
Note："-" represents that peak area is less than quantitative limit.
Comparative example
By the inventive method to 3 detections of lot number Shengmai Yin (Radix Codonopsis side) sample of same manufacturing enterprise and traditional immunization parent
Contrasted with post method, the preparation method of immune affinity column method need testing solution is：Precision takes Shengmai Yin (Radix Codonopsis side) 10mL, will
Solution then uses pure water 20mL drip washing, again with methanol 1mL wash-outs at the uniform velocity by the immune affinity column for having activated.Loading, drip washing and
Flow velocity 2mL/min during wash-out, eluent filtering, obtains final product.Immune affinity column method is with the inventive method gained need testing solution through height
Effect liquid phase chromatogram detection measured result is shown in Table 3.
The Magnetic solid phases extraction of table 3 and immune affinity column method determine knot to aflatoxin content in Shengmai Yin (Radix Codonopsis side)
Really (ng/mL)
Contrasted more than, what the method for the invention and Pharmacopoeia of People's Republic of China version in 2015 were recorded exempts from
Epidemic disease affinity column method measured result does not have notable difference, illustrates that the method for the invention measurement result is accurate, reliable.
Claims (10)
1. it is a kind of to determine aflatoxins B in Chinese patent drug simultaneously1, B2The method of content, it is characterised in that described method is：
(1) need testing solution is prepared
Take in Chinese patent drug addition pure water to be measured, ultrasonic disperse is uniform, pH is to 2~5 for regulation, is subsequently adding magnetic nanoparticle, stirs
Mix absorption and obtain mixed liquor；Gained mixed liquor is acted on through externally-applied magnetic field and is layered, and is inclined except supernatant liquor, and two are added in lower sediment
Chloromethanes and acetone volume ratio 2：1 mixed solvent, stir desorption, after through externally-applied magnetic field act on be layered, collect upper strata it is clear
Liquid, after volatilizing naturally at room temperature, surplus materials absolute methanol redissolves, filter membrane, collects filtrate and obtains final product need testing solution；
The concentration range of gained need testing solution is：0.3~3.4ng surplus materials/mL absolute methanols；
Described magnetic nanoparticle is：Tetraethyl orthosilicate-APTES modification Fe 3 O 4 magnetic
Nano particle；
(2) need testing solution detection
The need testing solution for taking step (1) preparation carries out high performance liquid chromatography detection, obtains the high-efficient liquid phase color of need testing solution
Spectrogram；The testing conditions of the high performance liquid chromatography are：Carbon octadecylsilane chemically bonded silica with particle diameter≤5 μm is as chromatographic column
Filler；With acetonitrile, methyl alcohol, the aqueous formic acid of volumetric concentration 0.1% mixed liquor as mobile phase, flow velocity：0.8~1.2mL/
min；Column temperature：20~30 DEG C；Fluorescence detector；Number of theoretical plate presses aflatoxins B1Peak is calculated and should be not less than 3000；
In described mobile phase, acetonitrile, methyl alcohol, the percentage by volume of the aqueous formic acid three of volumetric concentration 0.1% are respectively
10%~25%, 10%~25%, 50%~80%；
The fluorescence detector excitation wavelength：360nm, launch wavelength：440nm；
(3) standard curve is set up
Weigh aflatoxins B1, B2Standard items, hybrid standard product solution is configured to methyl alcohol, takes different amounts of hybrid standard product molten
Liquid, is detected respectively under the conditions of with step (2) identical high performance liquid chromatography detection, obtains the height of hybrid standard product solution
Effect liquid phase chromatogram figure；With hybrid standard product solution Plays product aflatoxins B1, B2Respective sample introduction quality is abscissa, mixing
The high-efficient liquid phase chromatogram Plays product aflatoxins B of standard solution1, B2The peak area of respective absworption peak is ordinate, is painted
Standard curve processed, carries out linear regression, obtains aflatoxins B1, B2Respective standard curve and equation of linear regression；
In the hybrid standard product solution, standard items aflatoxins B1Concentration be 0.3~10ng/mL, standard items aflatoxins B2
Concentration be 0.03~5ng/mL；
(4) testing sample result is determined
By aflatoxins B in the high-efficient liquid phase chromatogram of step (2) need testing solution1, B2The peak area numerical value generation at respective absorption peak
Enter in the equation of linear regression of step (3) foundation, be calculated aflatoxins B in need testing solution1, B2Content, and then calculate
Go out aflatoxins B in Chinese patent drug to be measured1, B2Content.
2. as claimed in claim 1 a kind of while determining aflatoxins B in Chinese patent drug1, B2The method of content, it is characterised in that
In step (1), the volumetric usage of the pure water is calculated as 3~7mL/g with the quality of Chinese patent drug to be measured.
3. as claimed in claim 1 a kind of while determining aflatoxins B in Chinese patent drug1, B2The method of content, it is characterised in that
In step (1), described Chinese patent drug to be measured is solid pharmaceutical preparation, and the magnetic nanoparticle is with the mass ratio of Chinese patent drug to be measured
0.01~0.02: 1.
4. as claimed in claim 1 a kind of while determining aflatoxins B in Chinese patent drug1, B2The method of content, it is characterised in that
In step (1), described Chinese patent drug to be measured is liquid preparation, and the quality consumption of the magnetic nanoparticle is with Chinese patent drug to be measured
Volume is calculated as 10~20mg/mL.
5. as claimed in claim 1 a kind of while determining aflatoxins B in Chinese patent drug1, B2The method of content, it is characterised in that
In step (1), the time of the stirring and adsorbing is 5~15min.
6. as claimed in claim 1 a kind of while determining aflatoxins B in Chinese patent drug1, B2The method of content, it is characterised in that
In step (1), the dichloromethane and acetone volume ratio 2：The volumetric usage of 1 mixed solvent is in terms of the quality of Chinese patent drug to be measured
It is 0.3~1.8mL/g.
7. as claimed in claim 1 a kind of while determining aflatoxins B in Chinese patent drug1, B2The method of content, it is characterised in that
In step (1), the time of the stirring desorption is 2~10min.
8. as claimed in claim 1 a kind of while determining aflatoxins B in Chinese patent drug1, B2The method of content, it is characterised in that
In step (2), in described mobile phase, acetonitrile, methyl alcohol, the percentage by volume point of the aqueous formic acid three of volumetric concentration 0.1%
Wei 20%, 20%, 60%.
9. as claimed in claim 1 a kind of while determining aflatoxins B in Chinese patent drug1, B2The method of content, it is characterised in that
In step (2), described chromatographic column is C18Post, 4.6mm × 250mm, the C18Filler carbon octadecylsilane bonding in post
The particle diameter of silica gel is 5 μm or 3.5 μm.
10. as claimed in claim 1 a kind of while determining aflatoxins B in Chinese patent drug1, B2The method of content, its feature exists
In described tetraethyl orthosilicate-APTES modification ferroferric oxide magnetic nanoparticle is by such as lower section
Method is prepared：
A () is by FeCl3·6H2O、FeCl2·4H2O mixes with deionized water, and under nitrogen protection, 85 DEG C of heating water baths are simultaneously stirred
30min, is then injected into ammoniacal liquor, then continue in 85 DEG C react 30min, be centrifuged afterwards, take precipitation successively with deionized water,
0.02mol/L sodium-chloride water solutions are washed, and 36h is dried at 40~60 DEG C, obtain ferroferric oxide magnetic nanoparticle；
The FeCl3·6H2O and FeCl2·4H2The mass ratio of O is 2~3：1；
The volumetric usage of the deionized water is with FeCl3·6H2O and FeCl2·4H2The gross mass of O is calculated as 12~13mL/g；
The volumetric usage of the ammoniacal liquor is with FeCl3·6H2O and FeCl2·4H2The gross mass of O is calculated as 1.4~1.8mL/g；
B step (a) gained ferroferric oxide magnetic nanoparticle is dissolved in methyl alcohol and pure water volume ratio 3~4 by ()：1 mixing
In solution, the ultrasound 10min under conditions of ultrasonic power is 600W is subsequently adding ammoniacal liquor, tetraethyl orthosilicate, is stirred at room temperature
10h, after be layered through externally-applied magnetic field, incline except supernatant, collect precipitation and be placed in 40~60 DEG C of vacuum drying chambers and dry 24h, grind
TEOS-MNPs magnetic nanoparticles are obtained after mill；
The volumetric usage of the methyl alcohol and pure water mixed solution is calculated as 200 with the quality of ferroferric oxide magnetic nanoparticle~
300mL/g；
The volumetric usage of the ammoniacal liquor is calculated as 20~30mL/g with the quality of ferroferric oxide magnetic nanoparticle；
The volumetric usage of the tetraethyl orthosilicate is calculated as 0.5~1.0mL/g with the quality of ferroferric oxide magnetic nanoparticle；
C () mixes step (b) gained TEOS-MNPs magnetic nanoparticles with APTES, toluene, rise
Temperature to 110 DEG C of backflow 10h, after be layered through externally-applied magnetic field, incline except supernatant, collect precipitation and be placed in 45~60 DEG C of vacuum drying chambers
In dry 24h, described APTES-TEOS-MNPs magnetic nanoparticles are obtained final product after grinding；
The volumetric usage of the APTES is calculated as 10 with the quality of TEOS-MNPs magnetic nanoparticles~
15mL/g；
The volumetric usage of the toluene is calculated as 100~150mL/g with the quality of TEOS-MNPs magnetic nanoparticles.
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