CN109142602A - A kind of preparation method of tetraodotoxin standard biological sample - Google Patents
A kind of preparation method of tetraodotoxin standard biological sample Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of tetraodotoxin standard biological sample.This preparation method is to extract basket whelk sample with acetic acid methanol solution, crude extract is purified with carbon nanotube solid-phase extraction column, the venom of purification carries out high-efficient liquid phase chromatogram purification, then freeze-drying concentration, crystallization is precipitated, obtains tetraodotoxin standard biological sample.The present invention from TTX extract, purifying and preparation sterling in terms of incision, using basket whelk as raw material, and by acetic acid methanol extraction, large volume carbon nanotube Solid phase extraction crude extract, after venom is concentrated, with prepare efficient liquid phase sample introduction carry out purifying preparation.Operation is easy and efficient with process for sample preparation methods of the present invention, prepares tetraodotoxin standard biological sample suitable for batch production, technology.
Description
Technical field
The present invention relates to a kind of preparation methods of tetraodotoxin standard biological sample.
Background technique
Fugutoxin (tetrodotoxin, TTX) is a kind of extremely strong non-protein class neurotoxin of toxicity, is present in river Puffer
(Tetraodontidae) and in other a variety of marine organisms bodies.It is as analgesic and anesthetic, and purposes is wide in medicine
General, aquatic livestock source field of food detection is also required to TTX standard items.Due to there is hardly possible in purification and technology of preparing and technique
Point, supply falls short of demand for the TTX standard items of high-purity.TTX preparation relies primarily on the method extracted from biomaterial and obtains, through excessive
The purifying of step and concentration step, to obtain the product of high-purity.
Traditional TTX standard sample preparation flow mostly using the internal organ of river Puffer fish as raw material is extracted, is extracted with acid flux material,
Impurity is removed by a variety of chromatography, the TTX product of high-purity is obtained in conjunction with repeated recrystallize and concentration step.Cui Jianzhou etc. is used
Aqueous acetic acid extracts the TTX in globefish liver, is carried out using two kinds of ion exchange resin of D201 and IR-86 to crude extract net
Change processing, Sephadex G-10 sephadex remove protein component, are isolated and purified, obtained pure to crude product with HPLC
The TTX product that degree is 99.5%.The country also has easy auspicious stove and Chen Weizhu by membrane separation technique, carries out sterling separation system with HPLC
It is standby, obtain the TTX product that purity is 99% and 97%.In addition, there are also utilize the thin of Shewanella alga and Bacillus fusiforms
Bacterium fermentation method obtains TTX product.This method can avoid the problem of largely catching and killing wild river Puffer because obtaining raw material, avoid simultaneously
It destroys, and is not limited by season and region caused by river Puffer resource and the ecological balance, but contained using the TTX that bacterium produces
It measures low, is difficult to large-scale application in actual production at present.
The chemical structure of TTX is more complicated (see attached drawing 1), and artificial synthesized there are larger difficulty, related at present artificial synthesized
The research of TTX technology is few.Japanese scholars Kishi is equal to 1972 has studied artificial synthesized racemic TTX for the first time, hereafter has again
The report of the artificial synthesis of stereoselective syntheses and asymmetric syntheses TTX occurs.
Summary of the invention
It is high-purity it is an object of the invention to be directed in existing TTX standard items preparation process to have the defects that technology and difficult point
The TTX standard items of degree the case where supply falls short of demand, a kind of preparation method of the tetraodotoxin biological standard sample of high-purity is provided, it should
Method preparation process with it is easy to operate, efficient, be applicable in tetraodotoxin basket whelk biological standard sample batch preparation and production.
The technical solution used in the present invention is:
A kind of preparation method of tetraodotoxin standard biological sample is to extract basket whelk sample with acetic acid methanol solution, will
Crude extract is purified with carbon nanotube solid-phase extraction column, and the venom of purification carries out high-efficient liquid phase chromatogram purification, is then freezed
Dry concentration, is precipitated crystallization, obtains tetraodotoxin standard biological sample.
The preparation method of this tetraodotoxin standard biological sample, comprising the following steps:
1) extraction of basket whelk sample: sufficiently homogeneous basket whelk sample of learning from else's experience is mixed with acetic acid methanol solution, ultrasonic water
Bath is extracted, then pipettes supernatant, and acetic acid methanol solution is added in residue, is repeated to extract 1 time, is merged crude extract twice,
It is spare；
2) carbon nanotube Solid Phase Extraction column purification: removing step 1) crude extract, with ammonium hydroxide adjust pH, pass through filling carbon
The Solid Phase Extraction column purification of nanotube, then extracts liquid in column, is successively eluted with methanol, pure water, extracts extraction column, then use second
Acid-acetonitrile solution elution, collects eluent, spare；
3) preparation liquid phase purifying: the eluent that step 2) is collected, water-bath rotary evaporation are concentrated into constancy of volume, remove second
Nitrile solvent；The venom of concentration is purified according to the condition of preparative high-performance liquid chromatographic sample introduction, according to going out for standard solution tetraodotoxin
Peak time determines target toxin component acquisition time in sample, collects the efflux in this time；Efflux rotary evaporation is dense
Contracting adjusts pH, freeze-drying to precipitation crystallization, i.e. tetraodotoxin standard biological sample with ammonium hydroxide.
In the step 1) of preparation method, the solid-liquid ratio of basket whelk sample and acetic acid methanol solution is 1: (5~6).
In the step 1) of preparation method, the volumetric concentration of acetic acid is 0.5%~1.5% in acetic acid methanol solution.
In the step 1) of preparation method, the condition of ultrasonic water bath extraction are as follows: ultrasonic power 150W~250W, ultrasonic wave frequency
Rate 5kHz~7kHz, 50 DEG C~70 DEG C of bath temperature, extraction time 10min~20min.
In the step 2) of preparation method, the solid-phase extraction column for loading carbon nanotube uses preceding activated processing, specifically: claim
The multi-walled carbon nanotube for taking 5g acidification uses methanol as dispersing agent, and wet process is loaded in empty 30mL pillar, sequentially adds
10mL acetic acidacetonitrile aqueous solution, the activation of 10mL ammonium hydroxide and balance pillar.
In the step 2) of preparation method, the volumetric concentration of acetic acid is 0.05%~0.15% in acetic acidacetonitrile aqueous solution, water
Volume ratio with acetonitrile is 8: 2；The volumetric concentration of ammonium hydroxide is 0.05%~0.15%.
The acidification of multi-walled carbon nanotube specifically: take multi-wall carbon nano-tube pipe powder to mix with sulfuric acid, nitric acid, ultrasound vibration
It swings, upper solution is removed after standing, rinsed repeatedly with water to neutrality, ground after dry, obtain the multi-wall carbon nano-tube of acidification
Pipe.
In the step 3) of preparation method, the condition of preparative high-performance liquid chromatographic sample introduction are as follows: chromatographic column: Inert Sustain
Amide prepares column, and specification is 20mm × 250mm, and diameter is 5 μm；Sample volume: 5mL；Flow velocity: 6.00mL/min；Column temperature: 35 DEG C,
Ultraviolet detection wavelength 200nm；Mobile phase A is acetonitrile, and Mobile phase B is 0.1% aqueous formic acid；Gradient elution program: 0~
10min, 10%~80% Mobile phase B；10.0min~12.5min, 80% Mobile phase B；12.5min~16.0min, 80%~
10% Mobile phase B；16.0min~25.0min, 10% Mobile phase B.
In the step 2) or step 3) of preparation method, adjusting pH with ammonium hydroxide is 8.5~9.0.
The beneficial effects of the present invention are:
The present invention is extracted from TTX, is cut in terms of purifying and preparation sterling, using basket whelk as raw material, and passes through acetic acid methanol
Extract, large volume carbon nanotube Solid phase extraction crude extract, after venom is concentrated, with preparation efficient liquid phase sample introduction purify
Preparation.Operation is easy and efficient with process for sample preparation methods of the present invention, prepares tetraodotoxin suitable for batch production, technology
Standard biological sample.
It is specific as follows:
(1) using toxicity, high, steady sources basket whelk replaces globefish ovary, liver etc. former as extracting to the method for the present invention
The carbon nanotube solid phase extraction column of filling certainly of material, acidified methanol ultrasound-assisted extraction high efficiency extraction toxin, large volume is effective
The venom of coarse extraction is purified, prepares liquid phase refined solution, vacuum freeze drying concentration, being made needs for detection and analysis and comparison
The tetraodotoxin standard biological sample wanted.
(2) present invention is by single factor experiment Optimized Extraction Process (solvent, solid-liquid ratio, extracting mode, temperature factor), certainly
Load the optimization of carbon nanotube Solid Phase Extraction large volume purification condition (carbon nanotube acidification processing, pillar filler amount of fill, solid phase extraction
Take purification process), preparation liquid phase purifying process research (chromatography gradient separations condition, optimization toxin efflux collect parameter) is built
A kind of preparation method of tetraodotoxin standard biological sample is found.Standard sample preparation process is simple, efficient, and batch is suitble to prepare
It is produced with technology.
Detailed description of the invention
Fig. 1 is TTX structural formula figure；
Fig. 2 is that the recovery rate of different extractants compares figure；
Fig. 3 be in methanol proportion of acetic acid to the influence curve figure of TTX extraction effect；
Fig. 4 is the curve graph of TTX recovery rate in sample under different temperatures；
Fig. 5 is influence curve figure of the sample solution difference pH value to the rate of recovery；
Fig. 6 is that the desorption effect of different ratio eluent compares figure；
Fig. 7 is TTX standard solution chromatogram.
Specific embodiment
A kind of preparation method of tetraodotoxin standard biological sample is to extract basket whelk sample with acetic acid methanol solution, will
Crude extract is purified with carbon nanotube solid-phase extraction column, and the venom of purification carries out high-efficient liquid phase chromatogram purification, is then freezed
Dry concentration, is precipitated crystallization, obtains tetraodotoxin standard biological sample.
The preparation method of this tetraodotoxin standard biological sample, comprising the following steps:
1) extraction of basket whelk sample: sufficiently homogeneous basket whelk sample of learning from else's experience is mixed with acetic acid methanol solution, ultrasonic water
Bath is extracted, then pipettes supernatant, and acetic acid methanol solution is added in residue, is repeated to extract 1 time, is merged crude extract twice,
It is spare；
2) carbon nanotube Solid Phase Extraction column purification: removing step 1) crude extract, with ammonium hydroxide adjust pH, pass through filling carbon
The Solid Phase Extraction column purification of nanotube, then extracts liquid in column, is successively eluted with methanol, pure water, extracts extraction column, then use second
Acid-acetonitrile solution elution, collects eluent, spare；
3) preparation liquid phase purifying: the eluent that step 2) is collected, water-bath rotary evaporation are concentrated into constancy of volume, remove second
Nitrile solvent；The venom of concentration is purified according to the condition of preparative high-performance liquid chromatographic (HPLC) sample introduction, according to standard solution fugutoxin
The appearance time of element, determines target toxin component acquisition time in sample, collects the efflux in this time；Efflux rotation
It is concentrated by evaporation, adjusts pH, freeze-drying to precipitation crystallization, i.e. tetraodotoxin standard biological sample with ammonium hydroxide.
Preferably, in the step 1) of preparation method, the solid-liquid ratio of basket whelk sample and acetic acid methanol solution is 1: (5~6)；
It is further preferred that the solid-liquid ratio of basket whelk sample and acetic acid methanol solution is 1: 5；Basket whelk sample and acetic acid methanol solution
Solid-liquid ratio refers to the quality of basket whelk sample and the volume ratio of acetic acid methanol solution.
Preferably, in the step 1) of preparation method, the volumetric concentration of acetic acid is 0.5%~1.5% in acetic acid methanol solution；
It is further preferred that the volumetric concentration of acetic acid is 1.0% in acetic acid methanol solution.
Further, the preparation method of 1% acetic acid methanol solution are as follows: 50mL acetic acid is mixed with 4.95L methanol, 4 DEG C of preservations
It is spare.
Preferably, in the step 1) of preparation method, the condition of ultrasonic water bath extraction are as follows: ultrasonic power 150W~250W,
Ultrasonic frequency 5kHz~7kHz, 50 DEG C~70 DEG C of bath temperature, extraction time 10min~20min；It is further preferred that super
The condition that sound water-bath is extracted are as follows: ultrasonic power 200W, ultrasonic frequency 6kHz, 60 DEG C of bath temperature, extraction time 15min.
Preferably, in the step 2) of preparation method, the crude extract volume of the solid-phase extraction column loading of carbon nanotube is loaded
No more than 200mL；Elute 8~12 times that acetic acidacetonitrile amount of aqueous solution used used is bed volume；It is further preferred that filling
The crude extract volume of the solid-phase extraction column loading of carbon nanotube is 200mL；Eluting acetic acidacetonitrile amount of aqueous solution used used is column
10 times of bed volume.
Preferably, in the step 2) of preparation method, the solid-phase extraction column for loading carbon nanotube uses preceding activated processing, tool
Body are as follows: the multi-walled carbon nanotube for weighing 5g acidification uses methanol as dispersing agent, and wet process is loaded in empty 30mL pillar,
Sequentially add 10mL acetic acidacetonitrile aqueous solution, the activation of 10mL ammonium hydroxide and balance pillar；Further, consolidating for carbon nanotube is loaded
Mutually extraction column bottom and upper layer joint sheet guarantee that filled column bed is smooth, and passability is good.
Preferably, in the step 2) of preparation method, in acetic acidacetonitrile aqueous solution the volumetric concentration of acetic acid be 0.05%~
0.15%, the volume ratio of water and acetonitrile is 8:2；The volumetric concentration of ammonium hydroxide is 0.05%~0.15%；It is further preferred that second
The volumetric concentration of acetic acid is 0.1% in acid-acetonitrile solution, and the volume ratio of water and acetonitrile is 8:2；The volumetric concentration of ammonium hydroxide is
0.1%.
Further, the preparation method of 0.1% acetic acidacetonitrile aqueous solution (8: 2, V/V) are as follows: 200mL acetonitrile adds 1mL second
Acid is uniformly mixed, 4 DEG C save backup with pure water constant volume to 1000mL.
Further, the preparation method of 0.1% ammonium hydroxide are as follows: 500 μ L ammonium hydroxide are uniformly mixed, 4 with pure water constant volume to 500mL
It DEG C saves backup.
Preferably, the acidification of multi-walled carbon nanotube specifically: take multi-walled carbon nanotube (MWCNTs) powder and sulfuric acid,
Nitric acid mixes, sonic oscillation, removes upper solution after standing, is rinsed repeatedly with water to neutrality, grinds, obtained at acidification after dry
The multi-walled carbon nanotube of reason；Further, the acidification of multi-walled carbon nanotube specifically: MWCNTs powder is weighed, in single port
In flask, the dense H of 60mL is added2SO4, the dense HNO of 120mL3, sonic oscillation 1h removes upper solution after standing, uses distilled water repeatedly
Rinsing is ground after 80 DEG C of vacuum drying to neutrality.
Preferably, in the step 3) of preparation method, the temperature of eluent water-bath rotary evaporation concentration is 50 DEG C~70 DEG C.
Preferably, in the step 3) of preparation method, the condition of preparative high-performance liquid chromatographic sample introduction are as follows: chromatographic column: Inert
Sustain Amide prepares column, and specification is 20mm × 250mm, and diameter is 5 μm；Sample volume: 5mL；Flow velocity: 6.00mL/min；Column
Temperature: 35 DEG C, ultraviolet detection wavelength 200nm；Mobile phase A is acetonitrile, and Mobile phase B is 0.1% aqueous formic acid；Gradient elution journey
Sequence: 0~10min, 10%~80% Mobile phase B；10.0min~12.5min, 80% Mobile phase B；12.5min~16.0min,
80%~10% Mobile phase B；16.0min~25.0min, 10% Mobile phase B.
Preferably, in the step 2) or step 3) of preparation method, adjusting pH with ammonium hydroxide is 8.5~9.0.
Preferably, in the step 3) of preparation method, the temperature of freeze-drying is -80 DEG C~-85 DEG C.
After the tetraodotoxin standard biological sample dissolved dilution obtained by step 3), purity survey is carried out with UPLC-MS/MS
Setting analysis.
Further, solvent used in dissolved dilution tetraodotoxin standard biological sample is the aqueous solution of formic acid acetonitrile, first
The volumetric concentration of formic acid is 0.1% in the aqueous solution of sour acetonitrile, and the volume ratio of water and acetonitrile is 1: 1.
Further, UPLC-MS/MS analysis chromatographic column: Acquity UPLC@BEH Amide column (100mm × 2.1mm,
1.7μm)；Column temperature: 40 DEG C；Sample volume: 10 μ L；Flow velocity: 0.3mL/min；Mobile phase: mobile phase A is 0.1% aqueous formic acid,
Mobile phase B is acetonitrile；Gradient elution program: 0~1.0min, 5%~60% mobile phase A；1.0~3.0min, 60% mobile phase
A；3.0~3.2min, 95%~5% mobile phase A；3.2~5min, 5% mobile phase A.
Further, UPLC-MS/MS analyzes Mass Spectrometry Conditions are as follows: electrospray ionisation (ESI)；Scanning mode: negative ions are same
When scan；Ion source temperature: 150 DEG C；Capillary voltage: 3.0kV；Desolvation temperature: 400 DEG C；Desolventizing gas flow:
900L/h；Collision gas flow velocity: 0.15mL/min, cone hole backflow airflow amount: 150L/h；Monitoring mode: multiple-reaction monitoring
(multiple-period MRM)。
The contents of the present invention are described in further detail below by way of specific embodiment.These specific implementation cases
For explaining only the invention, it is not intended to limit the present invention.
1 instrument and reagent
Instrument: 2545 type preparation solutions are mutually equipped with 2489UV detector (Waters, US)；Acquity UPLC I-
The triple level four bars mass spectrographs of Class/Xevo TQS Ultra Performance Liquid Chromatography instrument series connection (are equipped with electric spray ion source and Masslynx
Data processing software, Waters, US)；ALPHA2-4LSC type vacuum freeze drier (German Christ company)；5810
Type desk centrifuge (German Eppendorf company)；MS3 vortex mixer (German IKA company)；N-EVAP nitrogen evaporator (the U.S.
Organomation company).
Reagent: TTX standard items (purity >=99.0%, sigma company of the U.S.), (analysis is pure, Guangzhou chemistry for acetic acid, formic acid
Chemical reagent work), acetonitrile, methanol etc. (being chromatographically pure, sigma company of the U.S.), ammonium hydroxide (analyzes pure, Guangzhou Chemical Reagent Factory), second
Acid, methanol (being chromatographically pure, sigma company of the U.S.), (Beijing Deco island science and technology is limited by 50 μm of multi-walled carbon nanotube CNT103
Company).
The acquisition of 2 samples and preparation
Basket whelk biological sample is acquired from Zhanjiang.4 DEG C of Refrigerated Transports of whole biological sample go back to laboratory, knit to jelly
Line spiral shell sample is cleaned multiple times with pure water, after washing away surface mud, breaks shell into pieces, takes all soft tissues, with homogeneous instrument
Carry out homogenization.Prepared sample is using preceding in -20 DEG C of freezen protectives.
3 Instrument measuring conditions
3.1 UPLC-MS/MS determination conditions
3.1.1 liquid phase chromatogram condition
Chromatographic column: Waters Acquity UPLC@BEH Amide (100mm × 2.1mm, 1.7 μm)；Mobile phase: acetonitrile
(A) and 0.1% formic acid solution (B)；Gradient elution program: 0~1.0min, 5%~60%B；1.0~3.0min, 60%B；3.0
~3.2min, 60%~5%B；3.2~5.0min, 5%B.Column temperature: 40 DEG C；Sample volume: 10 μ L；Flow velocity: 0.30mL/min.
3.1.2 Mass Spectrometry Conditions
Ion source: electrospray ionisation (ESI)；Monitoring mode: multiple-reaction monitoring (MRM)；Scanning mode: cation scanning；
Ion source temperature: 150 DEG C；Capillary voltage: 3.0kV；Desolvation temperature: 400 DEG C；Desolventizing gas flow: 900L/hr；It touches
Hit gas velocity: 0.2mL/min；Cone hole backflow airflow amount: 150L/hr.Parent ion, daughter ion and the specific mass spectrometry parameters of TTX are shown in
Table 1.
1 TTX mass spectrum of table acquires MRM parameter
| Compound | Ionization mode | Parent ion/(m/z) | Daughter ion/(m/z) | Orifice potential/(V) | Collision energy (eV) |
| TTX | ESI+ | 320.1 | 302.0*, 161.9 | 35 | 20,35 |
Note: band * is quota ion in table 1.
3.2 preparation liquid phase determination conditions
Chromatographic column: Inert Sustain Amide prepares column (20mm × 250mm, 5 μm)；Sample volume: 5mL；Flow velocity:
6.00mL/min；Column temperature: 35 DEG C, ultraviolet detection wavelength 200nm.Mobile phase A is acetonitrile, and Mobile phase B is that 0.1% formic acid is water-soluble
Liquid, gradient elution program: 0~10min, 10%~80%B；10.0~12.5min, 80%B；12.5~16.0min, 80%~
10%B；16.0~25.0min, 10%B.
4 sample extractions purify and prepare purification step
(1) extraction of basket whelk sample: accurately weighing through sufficiently homogeneous sample 200g, and in 2L beaker, 1L is added
1% acetic acid methanol solution, 60 DEG C of ultrasonic water bath ultrasonic extraction 15min, pipettes supernatant, and 1% acetic acid first of 1L is added in residue
Alcoholic solution repeats to extract 1 time, merges 2 extracting solutions, be settled to 2L.
(2) carbon nanotube solid phase extraction column purifies: weighing MWCNTs powder, in single-necked flask, it is dense that 100mL is added
H2SO4, the dense HNO of 200mL3, sonic oscillation 1h removes upper solution after standing, is washed to neutrality, 80 DEG C of dryings with distillation repeatedly
After grind.The multi-walled carbon nanotube for weighing 5g acidification uses methanol as dispersing agent, and wet process is loaded on empty 30mL pillar
In, 10mL0.1% acetic acidacetonitrile aqueous solution (8: 2, V/V), the activation of 10mL0.1% ammonium hydroxide and balance pillar are sequentially added, is extracted
Residual liquid in column.Removing step (1) coarse extraction venom 200mL, is added dropwise ammonium hydroxide and is sufficiently stirred in sample liquid, adjusts pH
It is 8.5, passes through the small column purification of carbon nanotube.Completion of the sample is extracted in column after liquid, is successively drenched with 30mL methanol, 30mL pure water
It washes, extracts pillar, eluted with 50mL0.1% acetic acidacetonitrile aqueous solution (8: 2, V/V).Eluent is collected, for purifying preparation step
With.
(3) preparation liquid phase purifying: the purification venom that step (2) is collected, 60 DEG C of water-bath concentrated by rotary evaporations to constancy of volume remove
Acetonitrile solvent.According to 3.2 preparation HPLC condition sample introduction purifying on the venom of step (3) concentration, when according to standard solution TTX appearance
Between, it determines target toxin component acquisition time in sample, collects the efflux in this time.The concentration of efflux rotary evaporation,
PH 8.5~9.0 is adjusted with ammonium hydroxide, carries out freeze-drying process, -81 DEG C of freeze-dryings to precipitation sterling crystallization.
The standard biological sample of preparation is dissolved with 0.1% formic acid-acetonitrile solution (1: 1, V/V) and constant volume, is diluted to conjunction
Suitable concentration, UPLC-MS/MS carry out purity testing analysis.
5 Study of optimization
(1) extraction process: carrying out experimental analysis to the Extraction solvent type of TTX sample, bath temperature, liquid-to-solid ratio factor,
Test comparison is carried out by water-bath combination ultrasound assisted extraction and traditional water bath extracting method simultaneously.
As shown in Fig. 2, pure methanol solution is poor to the extraction effect of TTX in basket whelk, and acetic acid aqueous solution and acetic acid
Methanol solution is unobvious to the extraction efficiency difference of TTX.Acetic acid methanol solution wears biological tissue samples as Extraction solvent
Saturating ability is strong, settles the protein in sample, and uses acetic acid water can be by the part aqueous egg in sample when extracting
White matter and impurity extract, and pillar blocking is serious when subsequent purification operation, therefore selects acetic acid methanol solution as extraction
Solution.
Test compares the extraction effect of acetic acid methanol solution under different acetic acid volume ratios, and it is as shown in Fig. 3 to extract result.
When the volume fraction of the acetic acid in extracting solution is 0.5%~1%, with the raising of proportion of acetic acid, TTX in sample extracting solution
Concentration is continuously improved；When the volume fraction of the acetic acid in extracting solution is greater than 2~5%, obviously rising does not occur in extraction efficiency.
Therefore selecting concentration is that 1% acetic acid methanol solution extracts basket whelk, is operated convenient for subsequent purification.
It is extracted compared with traditional water bath heating means using water-bath combination ultrasonic wave added method, ultrasonic water bath method recovery rate improves
22.4%.When liquid-to-solid ratio is 1: 1,2: 1,3: Isosorbide-5-Nitrae: when 1, cannot sufficiently extract the TTX in basket whelk sample.Work as liquid-to-solid ratio
When 5: 1 and 6: 1, the TTX in sample can be effectively extracted.Consider that the extraction efficiency under 2 kinds of liquid-to-solid ratios is suitable, it is unnecessary to reduce
Reagent waste selects 5: 1 liquid-to-solid ratios to extract TTX in basket whelk sample.
Water bath heating temperature is improved, extraction efficiency can be increased, but excessively high Extracting temperature not only will increase the energy and disappear
Consumption, is also easy to make the other compositions in sample to be precipitated, and carrys out difficulty to subsequent sample purification work belt.20 DEG C, 30 DEG C, 40 DEG C,
The extract concentration of TTX is as shown in Fig. 4 in sample at 50 DEG C, 60 DEG C, 70 DEG C.As bath temperature changes, in sample
TTX recovery rate change, the TTX concentration in extracting solution and bath temperature, which exist, to be positively correlated.Bath temperature is 20 DEG C~60
DEG C when, the concentration of TTX is increased to 3.10 μ g/mL from 1.50 μ g/mL in extracting solution.When bath temperature rises to 70 DEG C, extracting solution
Concentration declines, it may be possible to which with the continuous raising of bath temperature, other coextraction ingredients in sample increase, and cause to extract
The extraction efficiency decline of TTX in liquid.It is thus determined that 60 DEG C are used as water-bath Extracting temperature.
Determine TTX extraction conditions in optimal basket whelk meat: using 1% acetic acid aqueous solution as extractant, using 60 DEG C as water
Bath temperature determines that solid-liquid ratio is 5: 1, and point 2 extractions pass through the TTX in water-bath combination ultrasound assisted extraction sample.
(2) purification process: the effective nitration mixture of multi-wall carbon nano-tube (nitric acid and sulfuric acid) is handled, on surface grafting hydroxyl,
The oxygen-containing polar group such as carboxyl, the activity for increasing carbon nanotube remove impurity.Multi-walled carbon nanotube adsorption capacity after acidification
By force, to the good purification of sample.When pillar specification is loaded in selection, the filling of 6mL sky pillar is selected, carbon nanotube is filled out after filling
Stock column bed has gap appearance, so that adsorption effect weakens, and adsorption capacity is limited, and purification process is easy leakage and wears, therefore selects
The SPE void column of 30mL carries out wet process filling.
The combination power of TTX and carbon nanotube filler is related with the pH of sample liquid is extracted, knot in acid or neutral pH range
Conjunction reserve capability is weaker, under the pH value to alkaline condition for needing to be adjusted sample liquid with ammonium hydroxide, can stablize with carbon nanotube filler and protect
Stay combination.By comparing the rate of recovery of extracting solution under condition of different pH, optimize the pH value of sample solution, sees attached drawing 5.Sample liquid is extracted to exist
Between pH 8.5~9.0 when loading, the adsorption binding energy power of filler is most strong, and as pH >=9.0, TTX is unstable, easy structure alienation
Or decompose, cause the TTX rate of recovery in extracting solution to decline, it is thus determined that the pH for extracting sample liquid is 8.5~9.0 to purify.
Test compares influence of the 2% acetic acid acetonitrile solution eluent of different ratio to TTX elution effect.Wherein 2%
Acetic acid acetonitrile solution and 2% acetic acidacetonitrile aqueous solution (20: 80, V/V) elute poor effect, can hardly wash the TTX of absorption
It takes off；2% acetic acidacetonitrile aqueous solution (40: 60, V/V) and 2% acetic acidacetonitrile aqueous solution (60: 40, V/V) are although eluent
Eluting peak shifts to an earlier date, and peak value occurs in 3~4BV, but the TTX total content in eluent is too low.Comprehensively consider, elution solution is selected
2% acetic acidacetonitrile aqueous solution (80: 20), amount of eluent 10BV are more appropriate.When elution, with 0.1% acetic acidacetonitrile water of 50mL
Solution (8: 2, V/V) can elute completely TTX.The desorption effect of different ratio eluent compares the visible attached drawing 6 of figure.
(3) purifying process is prepared: by prepared 10 μ g/mL TTX standard solution through preparation HPLC analysis, liquid chromatogram
Figure is as shown in Fig. 7.TTX appearance time is 8.996min, and object appearance since 8.20min terminates in about 1.5min
Appearance, it is thus determined that collecting the efflux in this time using 8.40~10.40min as target toxin component acquisition time.
Acquisition parameter is set with Waters Fraction Collector, efflux is acquired, 540mL is obtained
Refined solution.Obtained 540mL efflux will be collected and be concentrated into constant volume (removing acetonitrile solvent) by rotary evaporation, use ammonium hydroxide
PH 8.5~9.0 is adjusted, freeze-drying process is carried out, obtains sterling.Sterling is white crystal, odorless, is dissolvable in water acid molten
In liquid.
The sterling that 1.00mg is prepared is accurately weighed, is dissolved with 0.1% formic acid-acetonitrile solution (1: 1, V/V) and fixed
Hold to 10mL, be diluted to suitable concentration, carries out UPLC-MS/MS analysis, the purity > 99.0% of the standardized product of preparation.
Claims (10)
1. a kind of preparation method of tetraodotoxin standard biological sample, it is characterised in that: extract basket whelk with acetic acid methanol solution
Sample purifies crude extract with carbon nanotube solid-phase extraction column, and the venom of purification carries out high-efficient liquid phase chromatogram purification, so
Freeze-drying concentration afterwards is precipitated crystallization, obtains tetraodotoxin standard biological sample.
2. a kind of preparation method of tetraodotoxin standard biological sample according to claim 1, it is characterised in that: including with
Lower step:
1) extraction of basket whelk sample: sufficiently homogeneous basket whelk sample of learning from else's experience is mixed with acetic acid methanol solution, and ultrasonic water bath mentions
It takes, then pipettes supernatant, acetic acid methanol solution is added in residue, repeat to extract 1 time, merge crude extract twice, it is spare；
2) carbon nanotube Solid Phase Extraction column purification: removing step 1) crude extract, with ammonium hydroxide adjust pH, pass through filling carbon nanometer
The Solid Phase Extraction column purification of pipe, then extracts liquid in column, is successively eluted with methanol, pure water, extracts extraction column, then with acetic acid-
Eluent is collected in acetonitrile solution elution, spare；
3) preparation liquid phase purifying: the eluent that step 2) is collected, water-bath rotary evaporation are concentrated into constancy of volume, and it is molten to remove acetonitrile
Agent；The venom of concentration is purified according to the condition of preparative high-performance liquid chromatographic sample introduction, when according to the appearance of standard solution tetraodotoxin
Between, it determines target toxin component acquisition time in sample, collects the efflux in this time；The concentration of efflux rotary evaporation,
PH, freeze-drying to precipitation crystallization, i.e. tetraodotoxin standard biological sample are adjusted with ammonium hydroxide.
3. a kind of preparation method of tetraodotoxin standard biological sample according to claim 2, it is characterised in that: step 1)
In, the solid-liquid ratio of basket whelk sample and acetic acid methanol solution is 1: (5~6).
4. a kind of preparation method of tetraodotoxin standard biological sample according to claim 2 or 3, it is characterised in that: step
It is rapid 1) in, in acetic acid methanol solution the volumetric concentration of acetic acid be 0.5%~1.5%.
5. a kind of preparation method of tetraodotoxin standard biological sample according to claim 2, it is characterised in that: step 1)
In, the condition of ultrasonic water bath extraction are as follows: ultrasonic power 150W~250W, ultrasonic frequency 5kHz~7kHz, bath temperature 50
DEG C~70 DEG C, extraction time 10min~20min.
6. a kind of preparation method of tetraodotoxin standard biological sample according to claim 2, it is characterised in that: step 2)
In, the solid-phase extraction column for loading carbon nanotube uses preceding activated processing, specifically: weigh the multi-wall carbon nano-tube of 5g acidification
Pipe, uses methanol as dispersing agent, and wet process is loaded in empty 30mL pillar, sequentially adds 10mL acetic acidacetonitrile aqueous solution, 10mL
Ammonium hydroxide activation and balance pillar.
7. a kind of preparation method of tetraodotoxin standard biological sample according to claim 6, it is characterised in that: step 2)
In, the volumetric concentration of acetic acid is 0.05%~0.15% in acetic acidacetonitrile aqueous solution, and the volume ratio of water and acetonitrile is 8:2；Ammonium hydroxide
Volumetric concentration be 0.05%~0.15%.
8. a kind of preparation method of tetraodotoxin standard biological sample according to claim 6, it is characterised in that: multi wall carbon
The acidification of nanotube specifically: take multi-wall carbon nano-tube pipe powder to mix with sulfuric acid, nitric acid, sonic oscillation removes after standing
Upper solution is rinsed to neutrality with water repeatedly, grinds after dry, obtain the multi-walled carbon nanotube of acidification.
9. a kind of preparation method of tetraodotoxin standard biological sample according to claim 2, it is characterised in that: step 3)
In, the condition of preparative high-performance liquid chromatographic sample introduction are as follows: chromatographic column: Inert Sustain Amide prepares column, specification be 20mm ×
250mm, diameter are 5 μm；Sample volume: 5mL；Flow velocity: 6.00mL/min；Column temperature: 35 DEG C, ultraviolet detection wavelength 200nm；Mobile phase
A is acetonitrile, and Mobile phase B is 0.1% aqueous formic acid；Gradient elution program: 0~10min, 10%~80% Mobile phase B；
10.0min~12.5min, 80% Mobile phase B；12.5min~16.0min, 80%~10% Mobile phase B；16.0min~
25.0min, 10% Mobile phase B.
10. a kind of preparation method of tetraodotoxin standard biological sample according to claim 2 or 7, it is characterised in that: step
Rapid 2) or in step 3), adjusting pH with ammonium hydroxide is 8.5~9.0.
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