CN109001333A - The method that Liquid Chromatography-Tandem Mass Spectrometry measures 3 kinds of short and naked dinoflagellate toxins in shellfish - Google Patents
The method that Liquid Chromatography-Tandem Mass Spectrometry measures 3 kinds of short and naked dinoflagellate toxins in shellfish Download PDFInfo
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Abstract
The invention belongs to toxin detection technology fields.The invention discloses the methods of 3 kinds of short and naked dinoflagellate toxins in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish, optimize pre-treatment step in terms of extraction reagent, solid-phase extraction column, sample solution, eluent four, establishes the high performance liquid chromatography-tandem mass detection method of short and naked dinoflagellate toxin in shellfish.Pre-treating method in the present invention is simple, high sensitivity, and stability is good, it is measured while 3 kinds of short and naked dinoflagellate toxins suitable for shellfish, 3 kinds of short and naked dinoflagellate toxins in detection method Liquid Chromatography-Tandem Mass Spectrometry quantitative analysis shellfish in the present invention, the rate of recovery is high, reproducible, quantitative limit reaches 3.0 μ g/kg, method is reliable and stable, high sensitivity, meets at present to the analysis detection needs of short and naked dinoflagellate toxin, with application value, be conducive to investigating and following the trail for marine eco-environment monitoring and pollution sources.
Description
Technical field
The present invention relates to toxin detection technology fields, more particularly, in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish 3
The method of kind short and naked dinoflagellate toxin.
Background technique
Short and naked dinoflagellate toxin (Brevetoxin, BTX) is the fat-soluble algae toxin of a kind of polyethers, mainly by double whip first
The red-tide algae toxin that algae and Gymnodinium part algae generate.Short and naked dinoflagellate toxin be made of 10 to 11 cyclic structures it is big
Ring polyether substance is stronger sodium channel activation toxin, can open on excitation membrane in conjunction with the receptor target area VI of sodium channel
Sodium channel, cell membrane can be made to enhance the permeability of sodium ion, voltage-gated sodium channel is activated, generate stronger cell and go
Polarization causes the conduction of neuromuscular excitement to change, therefore also referred to as nerve saxitoxin.Short and naked dinoflagellate toxin
With thermal stability, make its elimination long half time in the organisms such as shellfish up to the dozens of days even several months, heating, microwave etc. are normal
Rule processing method cause toxin concentration higher because reducing the water content in aquatic products and cause consumer generate such as nausea,
The poisoning symptoms such as vomiting, diarrhea, paralysis, stupor.Red tide occurs for Area of The East China Sea in July sea area of Wenzhou within 2014, by this sea area
Shellfish samples investigation and analysis, find sea area in shellfish samples short and naked dinoflagellate toxin content be 1.23-10.41 μ g/kg；2017
Ningbo of Zhejiang in June sea area, which has been broken out, makes human health although content is not high by the toxic red tide of sociales of Gymnodinium breve
At potential hazard, it is necessary to reinforce the detection work of short and naked dinoflagellate toxin in aquatic products, in favor of monitoring the marine eco-environment and
Ensure fish quality.
Currently, the analytic approach of short and naked dinoflagellate toxin mainly has Mouse bioassay, cytotoxicity experiment, radioimmunology, enzyme
Linked immunosorbent assay and liquid chromatography tandem mass spectrometry.Wherein, Mouse bioassay is earliest, the most widely used analysis method of development,
But can not accurate quantification toxin content；Though enzyme-linked immunization detection time is short, easily there is cross reaction；And high-efficient liquid phase color
The chromatogram separating capacity of tandem mass spectrometry combination high performance liquid chromatography and the qualitative, quantitative ability of mass spectrum high sensitivity are composed, inspection is become
Survey the prefered method of analysis.Shellfish usually also causes containing secretion mucus other than containing impurity such as fat, protein, pigments
The phenomenon that often being emulsified in Liquid-liquid Extraction Processes, while a part of object is also adsorbed, cause the situation that the rate of recovery is low.
Summary of the invention
Many and diverse to solve detection method step existing in the prior art, detection sensitivity is too low and can not detect simultaneously
Detection efficiency and detection sensitivity can be improved the present invention provides one kind and at the same time energy in the problem of 3 kinds of short and naked dinoflagellate toxins
Enough detect the method for 3 kinds of short and naked dinoflagellate toxins in the Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish of 3 kinds of short and naked dinoflagellate toxins.
To achieve the above object, The technical solution adopted by the invention is as follows:
The method that Liquid Chromatography-Tandem Mass Spectrometry measures 3 kinds of short and naked dinoflagellate toxins in shellfish, comprising the following steps:
A) it extracts: weighing the sample to be tested of 1.00g, be placed in centrifuge tube A, addition 4mL organic extractant, vortex oscillation 2 minutes,
Room temperature sonic oscillation 10 minutes, 5000rpm revolving speed was centrifuged 6 minutes, and then supernatant is transferred in centrifuge tube B, toward centrifuge tube B
Middle addition 16mL water, sonic oscillation 0.5 minute, is made mixed liquor to be clean；
B) mixed liquor to be clean is added to the C for having used 10mL activating solution to activate18Solid-phase extraction column after end of the sample, uses 5mL
Leacheate elution processing, drains Liquid Residue in column and abandons whole effluxes, then eluted with 3mL eluent, with centrifuge tube C
Eluent is collected, vortex oscillation 1 minute, through organic phase filtering with microporous membrane, prepare liquid is made；
C) analysis measurement: short to 3 kinds in prepare liquid in the knit stitch of ultra performance liquid chromatography-tandem mass spectrum multiple-reaction monitoring pattern
Naked dinoflagellate toxin carries out qualitative and quantitative analysis.
Preferably, in step c) analysis measurement,
Liquid chromatogram test condition is as follows: chromatographic column is ACQUITYTM UPLC BEH C18Column (2.1mm × 100mm, 1.7 μ
M), 10 μ L of sampling volume, 4 DEG C of sample room temperature, 40 DEG C of column temperature, flow velocity 0.2mL/min, mobile phase A is to contain 0.1% formic acid
5mmol/L ammonium acetate solution, B is acetonitrile；Gradient elution: 0~0.5min, 50% mobile phase A；0.5~1.5min, 50%
~35% mobile phase A；1.5~9.0min, 35% mobile phase A；9.0~10min, 35%~50% mobile phase A；10~
11min, 50% mobile phase A；
Mass Spectrometer Method condition is as follows: electric spray ion source, cation scanning；Detection mode is multiple-reaction monitoring pattern；Capillary
Voltage is 3.0kV；Ion source temperature is 150 DEG C；Desolvation temperature is 450 DEG C；Taper hole throughput is 150L/h；Desolventizing gas
Temperature: 450 DEG C；Taper hole throughput: 150L/h；Desolventizing gas flow: 600L/h.
Preferably, sample to be tested is the shellfish samples to be measured after crushing is sufficiently homogeneous.
Preferably, organic extractant is one of acetone, methanol or acetonitrile.
Preferably, organic extractant is acetone.
Preferably, centrifuge tube A is the centrifuge tube of 15mL, centrifuge tube B is the centrifuge tube of 50mL.
Preferably, activating solution is the aqueous acetone solution of 20vol%.
Preferably, leacheate is the methanol aqueous solution of 20vol%.
Preferably, eluent is acetonitrile.
Preferably, the aperture of organic phase miillpore filter is 0.22 μm.
Therefore, the invention has the following advantages: the pre-treating method in the present invention is simple, high sensitivity, stability
It is good, it is measured while 3 kinds of short and naked dinoflagellate toxins suitable for shellfish；Detection method Liquid Chromatography-Tandem Mass Spectrometry in the present invention is fixed
3 kinds of short and naked dinoflagellate toxins in amount analysis shellfish, the rate of recovery is high, and reproducible, quantitative limit reaches 3.0 μ g/kg, and method stabilization can
It leans on, high sensitivity, meets at present to the analysis detection needs of short and naked dinoflagellate toxin, there is application value, advantageous Yu Haiyang
ECOLOGICAL ENVIRONMENTAL MONITORING and pollution sources are investigated and followed the trail.
Detailed description of the invention
Fig. 1 is the sample MRM figure that liquid-phase condition is not optimised；
Fig. 2 is the sample MRM figure after liquid-phase condition optimization；
Fig. 3 is BTXs corresponding response in different proportion acetonitrile；
Fig. 4 is BTXs Secondary ion Mass Spectrometry figure；
Fig. 5 is influence of the acetonitrile of different elution volumes to the short and naked dinoflagellate toxin rate of recovery；
Fig. 6 is that the MRM of 3.0 μ g/kg mark-on mussel samples schemes.
Specific embodiment
Further description of the technical solution of the present invention With reference to embodiment.
Obviously, the described embodiments are merely a part of the embodiments of the present invention, instead of all the embodiments.Based on this
Embodiment in invention, all other reality obtained by those of ordinary skill in the art without making creative efforts
Example is applied, shall fall within the protection scope of the present invention.
In the present invention, if not refering in particular to, all equipment and raw material is commercially available or the industry is common,
Method in following embodiments is unless otherwise instructed conventional method in that art.
Army uses following instrument and reagent in the determination and optimization process of following embodiment and test condition:
ACQUITYTM I-Class ultra performance liquid chromatography Xevo TQ-S mass spectrograph (Waters, US)；SPE-24 solid phase
Extraction equipment (supelco company of the U.S.)；MS3Digital eddy mixer (German IKA company)；Centrifuge5810 high
Fast centrifuge (German Eppendorf company)；Ultrasonic cleaner (Shanghai High Kudos Science Instrument Co., Ltd.)；N-EVAP-112
Nitrogen evaporator (Organomation company of the U.S.)；Sep-Pak Vac C18Solid-phase extraction column (6mL, 1000mg, U.S. Waters
Company).
BTX-A, BTX-B, BTX-C short and naked dinoflagellate toxin standard items (are 100 μ g, TaiWan, China ALGALCHEM is public
Department)；Formic acid, ammonium acetate (chromatographically pure, Sigma Co., USA)；Acetonitrile, methanol, acetone (chromatographically pure, German Merck company)；
Experimental water is the ultrapure water handled through Millipore system.
Short and naked dinoflagellate toxin hybrid standard stock solution (10 μ g/mL): short naked with methanol dissolution BTX-A, BTX-B, BTX-C
Ciguatoxin standard items, mixing are settled to 10mL, and -20 DEG C save, and validity period 6 months.
Short and naked dinoflagellate toxin hybrid standard uses liquid (1 μ g/mL): 1mL short and naked dinoflagellate toxin hybrid standard stock solution is pipetted,
It is made into 1 μ g/mL mixed solution with methanol dilution, -20 DEG C save, and validity period 1 month.
One, embodiment 1
A kind of method that Liquid Chromatography-Tandem Mass Spectrometry measures 3 kinds of short and naked dinoflagellate toxins in shellfish, specific steps are as follows:
A) sample to be tested for weighing 1.00g (being accurate to 0.01g) sufficiently homogeneous, is placed in 15mL centrifuge tube, and 4mL acetone is added,
Vortex oscillation 2min, room temperature sonic oscillation 10min, 5000r/min centrifugation 6min, is transferred to 50mL centrifuge tube for supernatant；It is past
16mL water is added in 50mL centrifuge tube, mixed liquor to be clean is made in sonic oscillation 0.5min；
B) it pipettes above-mentioned mixed liquor and adds to the C for having used 20% aqueous acetone solution of 10mL to activate18Solid-phase extraction column；End of the sample
Afterwards, it is eluted with 20% methanol aqueous solution of 5mL, drains Liquid Residue in column, and discard all of the above efflux, then with 3mL acetonitrile
Elution collects eluent with 15mL centrifuge tube, and vortex vibrates 1min and is made to be measured through 0.22 μm of organic phase filtering with microporous membrane
Liquid；
C) in the knit stitch of ultra performance liquid chromatography-tandem mass spectrum multiple-reaction monitoring pattern to 3 kinds of Gymnodinium breve poison in prepare liquid
Element carries out qualitative and quantitative analysis；
Wherein testing conditions are as follows:
Liquid chromatogram test condition is as follows: chromatographic column is ACQUITYTM UPLC BEH C18Column (2.1mm × 100mm, 1.7 μ
M), 10 μ L of sampling volume, 4 DEG C of sample room temperature, 40 DEG C of column temperature, flow velocity 0.2mL/min, mobile phase A is to contain 0.1% formic acid
5mmol/L ammonium acetate solution, B is acetonitrile；Gradient elution: 0~0.5min, 50% mobile phase A；0.5~1.5min, 50%
~35% mobile phase A；1.5~9.0min, 35% mobile phase A；9.0~10min, 35%~50% mobile phase A；10~
11min, 50% mobile phase A；
Mass Spectrometer Method condition is as follows: electric spray ion source, cation scanning；Detection mode is multiple-reaction monitoring pattern；Capillary
Voltage is 3.0kV；Ion source temperature is 150 DEG C；Desolvation temperature is 450 DEG C；Taper hole throughput is 150L/h；Desolventizing gas
Temperature: 450 DEG C；Taper hole throughput: 150L/h；Desolventizing gas flow: 600L/h；
The mass spectrums multiple-reaction monitoring experiment condition such as parent ion and daughter ion of short and naked dinoflagellate toxin is as shown in table 1.
The mass spectrometry parameters of 1 short and naked dinoflagellate toxin of table
* quota ion (Quantitative ion)
Two, the determination and optimization of test condition
1. liquid phase chromatogram condition optimizes
Tri- kinds of analyte structures of BTX-A, BTX-B, BTX-C are similar, belong to lipophilic compound, conventional C18Chromatographic column can be full
The needs of sufficient BTXs liquid-phase chromatographic analysis.Discovery when to BTXs standard solution progress chromatographic isolation, selects the 5 of 0.1% formic acid
Mmol/L ammonium acetate solution is as water phase, and acetonitrile is as organic phase, and 5min can efficiently separate three kinds of analytes, and peak shape is sharp
Symmetrically；But when analyzing with the same terms sample, occur that target peak can not effective integral, signal-to-noise ratio be low etc. that chromatographies are existing
As seeing Fig. 1.As seen from the figure, sample bring matrix effect makes baseline constantly raise extension, and it is one that miscellaneous peak and target peak, which mix,
Body can not separate.Extended for gentle Chromatogram Baseline, amplification miscellaneous peak and target peak chromatography differential separating effect using long chromatographic column
Analysis time runs low speed and the metastable liquid-phase condition of gradient, according to the separation of the steady situation, target peak of baseline
The information such as degree and signal-to-noise ratio, the best gradient of debugging optimization mobile phase.The effect adjusted through being compared gradient, comprehensive analysis duration,
It is final to determine the corresponding chromatographic column of experimental analysis and liquid-phase condition；With this condition, target peak can be in 11 minutes and miscellaneous peak
Separation, and peak type is sharp, baseline is steady, sees Fig. 2.
It generallys use initial flow phased soln analyte to remove solvent effect in liquid-phase chromatographic analysis field and is examined
It surveys；But BTXs belongs to fat-soluble toxins, and when analysis need to be dissolved in acetonitrile, and cannot use initial liquid phase.BTXs is not year-on-year
Corresponding response is shown in Fig. 3 in example acetonitrile, it is known that the corresponding BTXs response of 0~50% acetonitrile significantly increases；Response is in second
Nitrile is more than slowly to enhance after 50%, reaches maximum value when ratio is 100%.
2. the determination of mass spectral analysis condition
Compare the response signal of BTXs under positive and negative two ion mode in the source ESI respectively, and to taper hole temperature, desolventizing gas flow
Equal mass spectrometry parameters optimize.The result shows that: BTXs is in ESI+Response under mode is high compared with ESI- mode, can produce stable
[M+H]+Characteristic ion, determines it as the quasi-molecular ion peak of analyte, and optimizes capillary voltage and orifice potential.Pass through
Quasi-molecular ion peak second order ms cracking analysis, it is strong and weak according to the abundance of fragment ion, filter out quota ion and qualitative ion.
Since BTXs is the macrocyclic polyether substance that multiple cyclic structures are constituted as skeleton, the fragment ion after leading to cracking is less,
And qualitatively and quantitatively the stability and abundance of ion are strong no more than characteristic ion.As there is fragment ion in collision energy increase
(including qualitatively and quantitatively ion) decaying even disappear but characteristic ion there are still mass spectrum phenomenon, with Conventional compounds mass spectrum point
It analyses different.In view of the particularity of BTXs mass spectrum expression, by the optimization of the screening process of quantitative and qualitative ion and collision energy
Process is mutually linked, and records the abundance situation of each fragment ion under more different collision energies, final to determine that BTXs's is quantitative fixed
Property ion and collision energy, are shown in Table 1, Secondary ion Mass Spectrometry figure is shown in Fig. 4.
3. optimization for extracting condition
Short and naked dinoflagellate toxin belongs to fat-soluble algae toxin, and experiment is respectively compared acetone, methanol, acetonitrile and mentions to short and naked dinoflagellate toxin
Take efficiency.Methanol energy infiltrating tissues, precipitating proteins, but it is lower to the extraction efficiency of short and naked dinoflagellate toxin；Acetonitrile is that polarity is non-
Proton solvent has higher extracted rate to short and naked dinoflagellate toxin, but is more toxic；Acetone to the recovery rate of short and naked dinoflagellate toxin most
Height, and can be miscible by different proportion with water, be conducive to the solvent ratios for optimizing Solid Phase Extraction sample solution, bonding unit sample quality
Effective extraction volume, therefore select 4mL acetone as extractant.
4. the selection of solid-phase extraction column
Short and naked dinoflagellate toxin category polyethers toxoid, a large amount of existing hydrocarbon bond energys and C in structure18C-H bond in functional group
Nonpolar action power is generated, is suitble in C18Solid-phase extraction column enrichment purification.The nonpolar action power of extractant acetone is larger, is easy
It is combined with the functional group of solid-phase extraction column, causes the short and naked dinoflagellate toxin in acetone that cannot effectively be enriched with；Therefore by 4mL acetone
It is mixed with 16mL water as sample solution, to enhance the polarity of sample solution, weakens the nonpolar action power of sample solution, improve target
The bioaccumulation efficiency of object.20% methanol-water of experimental selection can not only effectively dispel the residual impurity in column as leacheate, also not shadow
Ring the chemical state of object.According to the polarity of eluent and object, acetonitrile, methanol is respectively adopted as eluent in experiment,
The evaluation indexes such as volume, the rate of recovery, matrix interference by investigation eluent select to be suitble to short and naked dinoflagellate toxin in C18Solid phase extraction
Take the eluent of post separation.The results showed that though methanol can effectively elute short and naked dinoflagellate toxin, its corresponding Chromatogram Baseline
Interfere larger, noise is relatively low；Acetonitrile can not only effectively elute object, and Chromatogram Baseline is more steady, and noise is relatively high, sensitivity
It is higher.Different volumes acetonitrile elutes the corresponding short and naked dinoflagellate toxin rate of recovery and sees Fig. 5, it is known that the acetonitrile of 3mL volume can be effective
Short and naked dinoflagellate toxin is eluted, realizes the high efficiency enrichment purification of short and naked dinoflagellate toxin in shellfish.
5. the quantitative limit and the range of linearity of method
Pipette appropriate BTXs hybrid standard using liquid compound concentration be 1.0 μ g/L, 5.0 μ g/L, 10.0 μ g/L, 20.0 μ g/L,
50.0 μ g/L, 100 μ g/L series standard working solutions, using BTXs concentration as abscissa, peak area is ordinate, and production standard is bent
Line.According to the instrument condition of this research, the linear relationship of three kinds of hexabromocyclododecane, the method range of linearity and recurrence side are obtained
Journey is shown in Table 2.The detection limit (LOD) for determining this method with 3 times of signal-to-noise ratio determines this method for 1.0 μ g/kg with 10 times of signal-to-noise ratio
Quantitative limit (LOQ) be 3.0 μ g/kg, as shown in Figure 6.
The linear equation and related coefficient of 2 short and naked dinoflagellate toxin of table
6. the method rate of recovery, precision, accuracy
It accurately weighs 1.00g feminine gender mussel, hang razor clam and clam sample, adds the BTXs of 5.0,20,50 tri- concentration levels of μ g/kg
The experiment of standard solution, each concentration repeats six times, and the calculation method rate of recovery and precision the results are shown in Table 3.The result shows that BTXs
It is the 5.0 μ g/kg of μ g/kg~50 in additive amount, average recovery rate is 80.0%~87.5%；Relative standard deviation be 1.32%~
5.40%.
The average recovery rate and precision (n=6) of 3 short and naked dinoflagellate toxin of table
7. conclusion
This method is using acetone as extractant, C18Solid phase extraction, it is 3 kinds short in Liquid Chromatography-Tandem Mass Spectrometry quantitative analysis shellfish
Naked dinoflagellate toxin, the rate of recovery is high, and reproducible, quantitative limit reaches 3.0 μ g/kg.Method is reliable and stable, and high sensitivity meets mesh
The preceding analysis detection to short and naked dinoflagellate toxin needs, and has application value, is conducive to marine eco-environment monitoring and pollution
It investigates and follows the trail in source.
It should be understood that those skilled in the art, can be improved or be become according to the above description
It changes, and all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
Claims (10)
1. the method that Liquid Chromatography-Tandem Mass Spectrometry measures 3 kinds of short and naked dinoflagellate toxins in shellfish, it is characterised in that the following steps are included:
A) it extracts: weighing the sample to be tested of 1.00g, be placed in centrifuge tube A, addition 4mL organic extractant, vortex oscillation 2 minutes,
Room temperature sonic oscillation 10 minutes, 5000rpm revolving speed was centrifuged 6 minutes, and then supernatant is transferred in centrifuge tube B, toward centrifuge tube B
Middle addition 16mL water, sonic oscillation 0.5 minute, is made mixed liquor to be clean；
B) mixed liquor to be clean is added to the C for having used 10mL activating solution to activate18Solid-phase extraction column after end of the sample, is drenched with 5mL
Washing lotion elution processing, drains Liquid Residue in column and abandons whole effluxes, then eluted with 3mL eluent, is received with centrifuge tube C
Collect eluent, vortex oscillation 1 minute, through organic phase filtering with microporous membrane, prepare liquid is made；
C) analysis measurement: short to 3 kinds in prepare liquid in the knit stitch of ultra performance liquid chromatography-tandem mass spectrum multiple-reaction monitoring pattern
Naked dinoflagellate toxin carries out qualitative and quantitative analysis.
2. the method for 3 kinds of short and naked dinoflagellate toxins in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish according to claim 1,
It is characterized by:
In the step c) analysis measurement,
Liquid chromatogram test condition is as follows: chromatographic column is ACQUITYTM UPLC BEH C18Column (2.1 mm × 100 mm, 1.7 μ
M), 10 μ L of sampling volume, 4 DEG C of sample room temperature, 40 DEG C of column temperature, 0.2 mL/min of flow velocity, mobile phase A is to contain 0.1% formic acid
5 mmol/L ammonium acetate solutions, B is acetonitrile；Gradient elution: 0~0.5 min, 50% mobile phase A；0.5~1.5 min, 50%
~35% mobile phase A；1.5~9.0 min, 35% mobile phase A；9.0~10 min, 35%~50% mobile phase A；10~11min,
50% mobile phase A；
Mass Spectrometer Method condition is as follows: electric spray ion source, cation scanning；Detection mode is multiple-reaction monitoring pattern；Capillary
Voltage is 3.0 kV；Ion source temperature is 150 DEG C；Desolvation temperature is 450 DEG C；Taper hole throughput is 150 L/h；Precipitation
Agent throughput is 600 L/h.
3. the method for 3 kinds of short and naked dinoflagellate toxins in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish according to claim 1,
It is characterized by: the sample to be tested is the shellfish samples to be measured after crushing is sufficiently homogeneous.
4. the method for 3 kinds of short and naked dinoflagellate toxins in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish according to claim 1,
It is characterized by: the organic extractant is one of acetone, methanol or acetonitrile.
5. the side of 3 kinds of short and naked dinoflagellate toxins in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish according to claim 1 or 4
Method, it is characterised in that: the organic extractant is acetone.
6. the method for 3 kinds of short and naked dinoflagellate toxins in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish according to claim 1,
It is characterized by: the centrifuge tube A is the centrifuge tube of 15mL, the centrifuge tube B is the centrifuge tube of 50mL.
7. the method for 3 kinds of short and naked dinoflagellate toxins in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish according to claim 1,
It is characterized by: the activating solution is the aqueous acetone solution of 20vol%.
8. the method for 3 kinds of short and naked dinoflagellate toxins in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish according to claim 1,
It is characterized by: the leacheate is the methanol aqueous solution of 20vol%.
9. the method for 3 kinds of short and naked dinoflagellate toxins in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish according to claim 1,
It is characterized by: the eluent is acetonitrile.
10. the side of 3 kinds of short and naked dinoflagellate toxins in a kind of Liquid Chromatography-Tandem Mass Spectrometry measurement shellfish according to claim 1
Method, it is characterised in that: the aperture of the organic phase miillpore filter is 0.22 μm.
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| CN110174470A (en) * | 2019-05-10 | 2019-08-27 | 宁波检验检疫科学技术研究院 | The high-flux detection method of marine biotoxins in a kind of aquatic products |
| CN113092610A (en) * | 2021-03-31 | 2021-07-09 | 中国海洋大学 | Method for extracting gymnodinotoxin from marine microalgae |
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