CN108872412A - QuEChERS method based on graphene establishes the UPLC-MS/MS detection method of fat-soluble saxitoxin - Google Patents
QuEChERS method based on graphene establishes the UPLC-MS/MS detection method of fat-soluble saxitoxin Download PDFInfo
- Publication number
- CN108872412A CN108872412A CN201810304789.8A CN201810304789A CN108872412A CN 108872412 A CN108872412 A CN 108872412A CN 201810304789 A CN201810304789 A CN 201810304789A CN 108872412 A CN108872412 A CN 108872412A
- Authority
- CN
- China
- Prior art keywords
- fat
- graphene
- uplc
- quechers
- saxitoxin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention relates to the UPLC-MS/MS detection methods that the QuEChERS method based on graphene establishes fat-soluble saxitoxin.The present invention establishes the UPLC-MS/MS quantitative detecting method of 9 kinds of fat-soluble saxitoxins in shellfish aquatic products：This method has many advantages, such as that quick, easy, efficient, the rate of recovery is high, organic solvent exposure is few, time saving；The QuEChERS Sample Pretreatment Technique of foundation, in conjunction with freezing grease removal technology and the high characterization of adsorption of graphene oxide, complex matrices (such as free fatty acid and pigment) can be effectively removed, obtain the detection rate of recovery effect of preferable fat-soluble saxitoxin, clean-up effect is good, cryogenic freezing technology auxiliary QuEChERS method is introduced to the clean-up effect of sample, matrix benefit, the sensitivity of improvement method can be substantially reduced；In conjunction with superelevation Liquid Chromatography-Tandem Mass Spectrometry technology, the detection of fat-soluble saxitoxin in shellfish samples is applied successfully, 9 kinds of fat-soluble saxitoxins can be detected simultaneously, dramatically increase 5 kinds than existing standard method, there is preferable applicability.
Description
Technical field
The present invention relates to the detection method of fat-soluble saxitoxin more particularly to a kind of reduction matrix benefit, improvement methods
Sensitivity the QuEChERS method based on graphene oxide establish fat-soluble saxitoxin UPLC-MS/MS multicomponent it is quantitative
Detection method.
Background technique
Marine biotoxins (saxitoxin) are refered in particular to mainly be generated by the toxic microalgae in ocean or microorganism, can be given birth in ocean
A big micromolecular poisonous chemical being enriched in object especially bivalve shellfish, to other biological including mankind's generation harm
Matter.For these biotoxins, researcher's initial stage is mainly classified as six major class according to caused poisoning symptom：Paralytic shellfish
Toxoid (Paralytic Shellfish Poisoning, PSP), research of diarrhetic shellfish poisons (Diarrhetic Shellfish
Poisoning, DSP), nerve saxitoxin (Neurotoxic Shellfish Poisoning, NSP), amnesia
Saxitoxin (Amnesic Shellfish Poisoning, ASP), west plus ichtyhotoxisin (Ciguatera Fish
Poisoning, CFP), bluish-green algae toxin.But go deep into research, new biotoxin type is constantly found, and more
Kind toxin such as OA is often formed with toxin AZA and PTX association, but has different mechanism of toxication, and original classification method cannot
Meets the needs of management and scientific research.Therefore, it 2004, is entrusted by FAO (Food and Agriculture Organization of the United Nation), the World Health Organization and inter-governmental ocean
Saxitoxin is divided into eight major class, respectively saxitoxin group by the Bivalve software biotoxin working group that member can be set up jointly
(Saxitoxin, STX), domoic acid group (Domoic acid, DA), okadaic acid toxin group (Okadaic acid,
OA), former more formic acid toxin groups (Azaspiracid, AZA), short and naked dinoflagellate toxin group (Brevetoxin, BTX), clam toxin group
(Pecenotoxins, PTX), Patinopecten yessoensis toxin group (Yessotoxin, YTX) and epimino toxoid (Cyclic
Imines, CIs).In addition to this, palytoxin (Palytoxins, P1TX) and west plus ichtyhotoxisin (Ciguatoxins, CTX)
It is being considered whether to draw and make in saxitoxin.
In existing 8 major class saxitoxin, STX and DA toxin group is relatively easily soluble in water, comparatively speaking OA, AZA, BTX, PTX,
YTX, CIs are polyether substance, have thermal stability, are easily soluble in the nonpolar organic reagent such as methanol, ether, therefore united
One is referred to as fat-soluble saxitoxin (lipophilic phycotoxins, LPs).The serious harm of saxitoxin has caused
The close attention of multiple countries, the developed country headed by American-European plus wait have formulated saxitoxin monitoring state plan in succession.It passes
The monitoring method of system be mainly periodically in related sea area bivalve shellfish carry out saxitoxin content and toxiferous algae type and
Quantity is monitored, to assess the risk of saxitoxin, to protection consumer safety, it is ensured that the sound development of shellfish industry is made
Positive contribution.However, traditional means, which need to acquire a large amount of shellfish and algae sample, could make saxitoxin effectively in advance
It is alert, a large amount of manpower and material resources are not only expended, but also timeliness is not strong.2004, MacKenzie et al. reported a kind of solid for the first time
Phase absorbing toxin tracer technique (Solid phase adsorption toxin tracking, SPATT) mainly utilizes spy
Anisotropic adsorbent material adsorbs target toxin, detects after then eluting, be concentrated and purifying in laboratory through LC-MS.SPATT
Technology is compared with the traditional method, and not only largely reduces sampling analysis workload, but also can be with monitoring objective sea area different water levels
The variation of saxitoxin has early warning effect to saxitoxin in combination with the situation of change of mussel poisoning element and toxic algae,
With very big development prospect.The main still Mouse bioassay of existing detection technique, there are animal welfare and detection limits for height
And the problems such as discrimination shellfish poison ingredient cannot be specified, although mass spectrum detection has also started the detection for saxitoxin, inspection
Survey that type is relatively simple or pre-treating method it is still necessary to further perfect.
Summary of the invention
It is an object of the invention to the Mass Spectrometry detection methods in order to solve to be currently used for saxitoxin, and it is more single to detect type
One defect and a kind of reduction matrix benefit is provided, the QuEChERS method based on graphene oxide of the sensitivity of improvement method is built
Found the UPLC-MS/MS multicomponent quantitative detecting method of fat-soluble saxitoxin.
To achieve the goals above, the present invention uses following technical scheme：
QuEChERS method based on graphene establishes the UPLC-MS/MS detection method of fat-soluble saxitoxin, the detection method
To extract shellfish samples by QuEChERS method, assist QuEChERS method to the clean-up effect of sample by cryogenic freezing technology,
Then by being analyzed into UPLC-MS/MS；Wherein purifying adsorbent uses graphene.
In the technical scheme, (1) uses graphene oxide as the purifying adsorbent of QuEChERS method for the first time, can get
The detection rate of recovery effect of preferable fat-soluble saxitoxin.(2) cryogenic freezing technology auxiliary QuEChERS method is introduced to sample
Clean-up effect, matrix benefit, the sensitivity of improvement method can be substantially reduced.(3) UPLC-MS/MS that method is established can be simultaneously
9 kinds of fat-soluble saxitoxins are detected, dramatically increase 5 kinds than existing standard method.
Preferably, the detection method the specific steps are：
1) QuEChERS method pre-treatment step：The good shellfish samples of 1g ± 0.01g homogeneous are accurately weighed in 15mL centrifuge tube, are added
Enter 2mL extracting solution, be vortexed concussion 30s；0.50g anhydrous magnesium sulfate is added, be vortexed concussion 1min, and 2.3 × 103G is centrifuged 5min, turns
Supernatant is moved in another 15mL centrifuge tube；It repeats to extract according to above-mentioned steps primary；Merge twice supernatant be placed in refrigerator-
20 DEG C of freezing 2h；Nitrogen at 40 DEG C of filtrate obtained is blown to close dry, addition 1mL by funnel of the rapid mistake equipped with absorbent cotton after taking-up
Extracting solution redissolves；Then 50.0mg graphene oxide and 100.0mg anhydrous magnesium sulfate is added, be vortexed concussion 5min, and 1.0 × 104g
It is centrifuged 5min, supernatant is analyzed after 0.22 μm of nylon leaching film filters into UPLC-MS/MS；
2) preparation of standard solution：According to the solubility and property of each toxin standard items, suitable toxin is drawn with pipettor
Standard items list mark, with methanol constant volume, so that the concentration of h-YTX, YTX are 400ng/mL, the concentration of OA, DTX1, DTX2, SPX are
200ng/mL；The concentration of AZA1, AZA2, AZA3 are the standard working solution of 50ng/mL, freezen protective under the conditions of -20 DEG C.
Preferably, mobile phase selective flow phase A：0.15% ammonium hydroxide and Mobile phase B：Methanol, the matter after 9 kinds of toxin optimizations
Composing parameter is：Ion source：ESI electric spray ion source, negative ions scan simultaneously；Detection mode：MRM multiple-reaction monitoring；Capillary
Tube voltage：ESI+：3.5kv；ESI-：3.0kv；Ion source temperature：145℃；Desolventizing temperature：450℃；Spray voltage：4000V；
Taper hole throughput：50L/h；Desolventizing gas flow：750L/h.
Preferably, UPLC-MS/MS chromatographic condition is：Chromatographic column：Waters XBridge C18 chromatographic column 100mm ×
2.1mm, 5.0 μm；Column temperature：40℃；Sample volume：10μL；Mobile phase：A：0.15% ammonium hydroxide；B：Methanol；Flow velocity：0.3mL/min；
Type of elution：Gradient elution.
Preferably, it is 7: 2: 1 methanol/ethanols/isopropanol that step 1) extracting solution, which is volume ratio,.
Preferably, purifying adsorbent is graphene oxide, reduced graphene, amination graphene or carboxyl graphite
Alkene.
The beneficial effects of the invention are as follows：
The present invention establishes the UPLC-MS/MS quantitative detecting method of 9 kinds of fat-soluble saxitoxins in shellfish aquatic products：This method
Have many advantages, such as that quick, easy, efficient, the rate of recovery is high, organic solvent exposure is few, time saving；
(1) the QuEChERS Sample Pretreatment Technique established, the high absorption in conjunction with freezing grease removal technology and graphene oxide are special
Property, it can effectively remove complex matrices (such as free fatty acid and pigment), the detection for obtaining preferable fat-soluble saxitoxin is returned
Yield effect, clean-up effect is good, introduces cryogenic freezing technology auxiliary QuEChERS method to the clean-up effect of sample, can significantly subtract
Few matrix benefit, the sensitivity of improvement method；
(2) the QuEChERS method after optimizing successfully is answered in conjunction with superelevation Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) technology
With the detection of saxitoxin fat-soluble in shellfish samples, 9 kinds of fat-soluble saxitoxins can be detected simultaneously, it is more aobvious than existing standard method
It writes and increases by 5 kinds, there is preferable applicability.
Detailed description of the invention
Fig. 1 be C18 of the present invention, PSA, GCB and GO as adsorbent when fat-soluble saxitoxin the rate of recovery.
Fig. 2 is graphene oxide, reduced graphene, amination graphene and carboxylated graphene of the present invention as adsorbent
When fat-soluble saxitoxin the rate of recovery.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further explained with attached drawing：
Laboratory apparatus
Waters superelevation liquid phase systems (ACQUITYUPLC) and triple quadrupole tandem mass spectrometers (XEVO-TQ), containing electron spray from
Component (ESI) and atmosphere pressure chemical ion source (APCI) (Waters, USA)；
Chromatographic column：Waters XBridge C18 column (5 μm, 100mmX2.1mm i.d.) Acquity HSS-T3
Column (1.8 μm, 100mm × 2.1mm i.d.) Atlantics HILIC Silica column (Waters, 150mm ×
2.1mm i.d., 5 μm) (Waters, MA, USA)；
Pure water meter：ELGA PURELAB Ultra+HitechRoDI plus pure water preparation instrument (ELGA, UK)；
Homogeneous instrument：PoMron (PT2000, Switzerland)；
Refrigerated centrifuge：Type 3K-18 (Sigma, Germany)；
Turbula shaker：SCILOGXE MX-S (SCILOGEX, USA)；
Tissue mincer：IKA T-18 (IKA, Germany)；
Assay balance：ELECTRONIC BALANCE (SHIMADZU, Japan)；
Nitrogen evaporator：N-EVAP-24 (Oragnic Association, USA)；
Ultrasonic washing instrument：Shanghai High Kudos Science Instrument Co., Ltd.；
Experiment reagent
Acetonitrile：HPLC grades, purity > 99.9%, it is purchased from Thermo Fisher Scientific, USA；
Methanol：HPLC grades, purity > 99.9%, it is purchased from TEDIA COMPANY.IN USA；
Isopropanol：HPLC grades, purity > 99.8%, it is purchased from TEDIA COMPANY.IN USA；
Acetone：HPLC grades, purity > 99.5%, it is purchased from TEDIA COMPANY.IN USA；
N-hexane：HPLC grades, purity > 99.5%, it is purchased from Honeywell, USA；
Ethyl alcohol：Pure, purity > 99.7% is analyzed, parent company of Chinese Medicine group is purchased from；
Ammonium hydroxide：Pure, purity 25%~28% is analyzed, parent company of Chinese Medicine group is purchased；
Ultrapure water：ELGA pure water meter is made, conductivity >=18.2M Ω；
Solid-phase extraction column：OASIS HLB is purchased from Waters, USA；
Cleanert C18, PSA, GCB are purchased from Agela Technologies, USA；
Graphene adsorbent material：Purchased from Nanoinnova technologies company (Madrid, Spain)；
Anhydrous magnesium sulfate (MgSO4), it is purchased from parent company of Chinese Medicine group；
9 kinds of normaltoxins (OA, DTX1, DTX2, YTX, h-YTX, SPX, AZA1, AZA2, AZA3) are purchased from Canadian country and grind
Jiu Yuan marine organisms research institute (National Research Council, Halifax, NS, Canada).
QuEChERS method based on graphene establishes the UPLC-MS/MS detection method of fat-soluble saxitoxin, the detection
Method is to extract shellfish samples by QuEChERS method, assists QuEChERS method to imitate the purification of sample by cryogenic freezing technology
Fruit, then by being analyzed into UPLC-MS/MS；Wherein purifying adsorbent uses graphene.
The detection method the specific steps are：
1) QuEChERS method pre-treatment step：The good shellfish samples of 1g ± 0.01g homogeneous are accurately weighed in 15mL centrifuge tube, are added
Enter 2mL extracting solution, methanol/ethanol/isopropanol of volume ratio 7: 2: 1, be vortexed concussion 30s；0.50g anhydrous magnesium sulfate is added,
Be vortexed concussion 1min, and 2.3 × 103G is centrifuged 5min, shifts supernatant in another 15mL centrifuge tube；It is repeated according to above-mentioned steps
It extracts primary；Merge supernatant twice and is placed in -20 DEG C of freezing 2h in refrigerator；Funnel of the rapid mistake equipped with absorbent cotton after taking-up, will
Nitrogen is blown to close dry at 40 DEG C of obtained filtrate, and 1mL extracting solution is added and redissolves；Then be added 50.0mg graphene oxide and
100.0mg anhydrous magnesium sulfate, be vortexed concussion 5min, and 1.0 × 104G is centrifuged 5min, and supernatant is filtered through 0.22 μm of nylon leaching film
Afterwards, it is analyzed into UPLC-MS/MS；
2) preparation of standard solution：According to the solubility and property of each toxin standard items, suitable toxin is drawn with pipettor
Standard items list mark, with methanol constant volume, so that the concentration of h-YTX, YTX are 400ng/mL, the concentration of OA, DTX1, DTX2, SPX are
200ng/mL；The concentration of AZA1, AZA2, AZA3 are the standard working solution of 50ng/mL, freezen protective under the conditions of -20 DEG C.
Purifying adsorbent is graphene oxide, reduced graphene, amination graphene or carboxylated graphene.
Shellfish samples are collected in Ningbo City, Zhejiang Province harbor area.The shellfish samples that acquisition comes are cleaned with clear water, are taken
Complete fresh soft tissue out, is put into tissue mashing machine and smashs to pieces, save under the conditions of obtained sample is put into -80 DEG C.
Signal interference when suitable liquid phase chromatogram condition is for improving chromatography selectivity and reducing Mass Spectrometer Method is very
It is necessary.Report based on forefathers, alkaline mobile phase would generally provide one for the detection of fat-soluble saxitoxin and more add
Selectivity, accuracy and the lower quantitative limit (LOQ) of beauty.In the present invention, basic mobile phase selects 0.15% ammonium hydroxide (stream
Dynamic phase A) and methanol (Mobile phase B), the part mass spectrometry parameters after 9 kinds of toxin optimize it is as follows：
Ion source：Electric spray ion source (ESI), negative ions scan simultaneously；
Detection mode：Multiple-reaction monitoring (MRM)；
Capillary voltage：ESI+：3.5kv；ESI-：3.0kv；
Ion source temperature：145℃；
Desolventizing temperature：450℃；
Spray voltage：4000V；
Taper hole throughput：50L/h；
Desolventizing gas flow：750L/h
The mass spectrums multiple-reaction monitoring condition such as orifice potential and collision energy is shown in Table 1.
Mass spectrum multiple-reaction monitoring (MRM) condition of the fat-soluble saxitoxin of table 1
The foundation of fat-soluble saxitoxin UPLC-MS/MS chromatographic condition
Chromatographic column：Waters XBridge C18 chromatographic column (100mm × 2.1mm, 5.0 μm)；
Column temperature：40℃；
Sample volume：10μL；
Mobile phase：A：0.15% ammonium hydroxide；B：Methanol；
Flow velocity：0.3mL/min；
Type of elution：Gradient elution；
Gradient elution program is shown in Table 2.
The gradient elution of 2 target saxitoxin of table
| Time (min) | Flow velocity (mL/min) | %A | %B | Curve |
| initial | 0.3 | 95 | 5 | 0 |
| 0.1 | 0.3 | 95 | 5 | 6 |
| 6 | 0.3 | 0 | 100 | 6 |
| 10 | 0.3 | 0 | 100 | 6 |
| 10.1 | 0.3 | 95 | 5 | 1 |
| 12 | 0.3 | 95 | 5 | 1 |
Methodology validation
The quantitative inspection of QuEChERS combination superelevation liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) the method detection saxitoxin of optimization
Surveying methodology validation includes following parameter：Linearly, matrix effect, accuracy, precision, detection limit (LOD) and quantitative limit
(LOQ).Since saxitoxin does not have suitable internal standard, external standard method is selected to be equipped with matrix-matched curve quantitative.Matrix matching
Curve negotiating bare substrate adds the saxitoxin hybrid standard product of known concentration, adds according to gradient concentration, bare substrate liquid
It is the optimized QuEChERS purification of shellfish samples crude extract.All experiments are repeated three times progress.Standard curve
Be linearly by related coefficient (r2) embody, in a few days repeated (Intra-day) and in the daytime reproducibility (Inter-day) and
The judgement of precision is judged by using the matrix mark-on measuring rate of recovery and relative standard deviation (RSD).Detection limit
(LOD) and quantitative limit (LOQ) is by matrix mark-on liquid sample detection, when signal-to-noise ratio (S/N) is 10: 1, as quantitative limit (LOQ)；
When to continue to be diluted to signal-to-noise ratio (S/N) be 3: 1, as method detection limit (LOD).
The optimizing research of Extraction solvent
Complex matrices ingredient and UPLC-ESI-MS/MS system in mussel extract are incompatible.Therefore, in order to obtain more
The sample preparation efficiency of high mass signal, the preferable rate of recovery and higher degree, by existing QuEChERS method into
Row improves, and makes it have higher extraction efficiency and relatively simple operation process.In general, using sample is extracted in solvent appropriate
Target analytes in product, this is the initial step of QuECHERS method.Therefore, methanol, ethyl alcohol, isopropanol, first are selected respectively
Alcohol: ethyl alcohol: isopropanol (7: 2: 1, v/v/v), methanol: ethyl alcohol: isopropanol (2: 7: 1, v/v/v), methanol: ethyl alcohol: isopropanol
(4.5: 4.5: 1, v/v/v) is used as Extraction solvent, and optimal extraction reagent is obtained by the rate of recovery of the saxitoxin of extraction.Knot
Fruit shows for from the point of view of most of fat-soluble seashells toxin, when selection methanol: ethyl alcohol: isopropanol (7: 2: 1, v/v/v)
When as extractant, the rate of recovery of 9 kinds of saxitoxins is all about 100%, smooth fluctuations, obtains the optimal rate of recovery.
The Selecting research of adsorbent
When matrix effect normally results in LC-MS/MS analysis, the enhancing or inhibition of fat-soluble saxitoxin mass signal.Therefore,
Shellfish samples carry out freezing processing after tentatively extracting immediately, in -20 DEG C of 2h arranged below in lead box, pay attention to adherent, make
Completely, then rapid filtration, removes most of lipid in shellfish samples for shellfish samples freezing.But the color of crude extract
There is no significant change occurs, show that the pigment in shellfish samples crude extract is not frozen step removing.In general, PSA, C18
It can be used for free fatty acid and pigment removal with GCB.Therefore, by comparing different types of adsorbent, including C18, PSA, GCB
And GO, assess its removal ability to interference component.The result shows that the clean-up effect relative to PSA and C18, uses GCB and GO
Carry out purified extracting solution more tend to it is transparent.It is moreover found that carrying out purified crude extract, matrix enhancement with C18 and PSA
Effect is obvious, this go out with document report in LC-MS/MS analysis, YTXs and AZAs are intended to letter by matrix enhancement effect
Number enhancing is consistent.In addition, although pigment removal effect when with GCB and GO is difficult when being purified using dispersive solid-phase extraction (d-SPE)
To distinguish, but GO adsorbent shows better pigment removal ability, because in the linear range, GO provides satisfactory
The rate of recovery, the rate of recovery between 82.3%-111.7%, relative standard deviation (RSD) be lower than 10.0%, as a result such as 3 institute of table
Show.
The rate of recovery and RSD of fat-soluble saxitoxin when table 3 C18, PSA, GCB and GO are as adsorbent
However, causing its rate of recovery lower, h-YTX since height of the GCB adsorbent to YTX and h-YTX retains in purification process
The rate of recovery with YTX is respectively 50.4% and 58.6%.The above results show the absorption for using GO as QuECHERS method
Agent obtains the preferable rate of recovery with the best ability for removing impurity in extracting solution, and rate of recovery range is 88.6%-
108.3%.As shown in Figure 1.
The graphene comparative studies of different shape
Investigate four kinds of graphenes --- graphene oxide, reduced graphene, amination graphene and carboxylated graphene conduct
Deimpurity ability is gone when QuECHERS method adsorbent, evaluation index is used as using the rate of recovery and relative standard deviation (RSD)
It is compared.As a result as shown in table 4, Fig. 2.In four kinds of graphenes, graphene oxide is the most stable for the absorption of target toxin；
Reduced graphene and amination graphene are higher for the reservation of h-YTX and YTX saxitoxin, cause the rate of recovery too low；Carboxylated
For graphene for graphite oxide, recycled in its entirety rate is below graphene oxide.Therefore, selective oxidation graphene is as net
Change adsorbent.
Table 4 utilizes graphene oxide, reduced graphene, rouge when amination graphene and carboxylated graphene are as adsorbent
The rate of recovery of dissolubility saxitoxin
The optimization of Mass Spectrometry Conditions
In order to provide better signal response when mass spectrum is quantitative, every kind of fat-soluble saxitoxin standard items first individually directly into
Sample, by multiple-reaction monitoring pattern (MRM) negative ions, scanning discovery, AZAs and SPX saxitoxin are suitable for cation mould simultaneously
Formula, YTXs, DTXs and OA saxitoxin are suitable for negative ion mode.Therefore, it when detecting 9 kinds of saxitoxins, needs in parent ion
Respond continuous conversion scan between highest daughter ion, response is used as quota ion compared with macroion in ion pair.To every
Kind analyte, optimizes the parameter of tandem mass spectrum (MS/MS), including orifice potential, collision voltage, quota ion and qualitative ion,
As shown in table 1.
The optimization of chromatographic condition
Signal interference has vital work when suitable chromatographic separation condition is for improving resolution ratio and reducing Mass Spectrometer Method
With.In general, alkaline mobile phase can provide better sensitivity, accuracy and lower quantitative limit and detection limit.In the present invention
In, mobile phase is 0.15% ammonium hydroxide (mobile phase A) and methanol (Mobile phase B), is optimized for fat-soluble shellfish by gradient elution
The separation of toxin provides enough resolution ratio.
Although QuEChERS method can effectively remove matrix components, so that higher accuracy is obtained, daily
In LC-MS/MS analysis, it can not avoid influencing brought by the matrix effect in sample completely.Interference component present in matrix
Matrix enhancement or substrate inhibition effect can be generated to target analytes, due to Gao Ling of the ion source ESI when tracking target substance
Sensitivity can cause target substance quantitatively to repeat or quantitatively lack.It therefore, in the present invention, is effectively matrix effect to be avoided to bring
Quantitative inaccuracy, matrix effect is evaluated using external standard method and matrix-matched curve.Meanwhile matrix effect can be because of base
Matter and the difference of saxitoxin and there is difference, therefore, in order to assess the stability of QuEChERS method, made a gift of using clam and purple
The two different matrix-matched curves of shellfish sample show (table 5) according to the slope of matrix-matched curve, based on GO and freezing grease removal
The QuEChERS method of step can substantially eliminate main matrix ingredient, and the stabilization of the linear and signal of this method is greatly improved
Property.Simultaneously, the results showed that, it can be used for the measurement of other shellfish samples using the matrix-matched curve of specific mussel extract.
Matrix effect evaluation table in 5 clam of table and Mytilus galloprovincialis
Linearly, LOD (detection limit) and LOQ (quantitative limit)
Handle to obtain object content to be respectively 5 blank sequences addition styles according to method in front of sample after optimization, then into
Row UPLC-MS/MS detection.Using the response area y of target analytes as ordinate, concentration X (μ g/kg) is added with target analytes
For abscissa, standard curve is drawn.As known from Table 6, in the matrix-matched curve of building, 9 kinds of fat-soluble saxitoxins：h-
YTX and YTX is 6.0-80.0 μ g/kg；DTX1, DTX2, OA and SPX are 3.0-40.0 μ g/kg；AZA1, AZA2, AZA3 are
0.75-10.0 μ g/kg is in a linear relationship.The result shows that related coefficient (r2) range be 0.9827~0.9997, this shows to test
Obtain satisfactory linear, the method after optimization has enough applicabilities, and it is quantitative to can satisfy fat-soluble saxitoxin
The analysis requirement of detection.Table 6 shows that method detection limit (LOD) is 0.10-1.47 μ g/kg, and quantitative limit (LOQ) is 0.32-4.92
μ g/kg is far below European Union's maximum residue limit (MRL).These statistics indicate that, this method have fabulous sensitivity.
The range of linearity of the fat-soluble saxitoxin of table 6, related coefficient (r2), detection limit (LOD) and quantitative limit (LOQ)
| Saxitoxin | The range of linearity/(μ g/kg) | Correlation coefficient r2 | Detection limit (μ g/kg) | Quantitative limit (μ g/kg) |
| h-YTX | 6-80 | 0.9936 | 1.47 | 4.92 |
| YTX | 6-80 | 0.9943 | 0.74 | 2.41 |
| DTX1 | 3-40 | 0.9930 | 0.66 | 2.19 |
| DTX2 | 3-40 | 0.9940 | 0.81 | 2.77 |
| OA | 3-40 | 0.9997 | 0.75 | 2.53 |
| AZA1 | 0.75-10 | 0.9827 | 0.17 | 0.55 |
| AZA2 | 0.75-10 | 0.9905 | 0.18 | 0.65 |
| AZA3 | 0.75-10 | 0.9990 | 0.13 | 0.43 |
| SPX1 | 3-40 | 0.9966 | 0.10 | 0.32 |
The rate of recovery and precision
Using shellfish as sample, 3 horizontal matrix mark-on experiments are done, is repeated 3 times respectively, saves the sample pre-treatments by 2.2.2
Method carries out the rate of recovery and precision test.7 be the results are shown in Table it is found that 9 kinds of fat-soluble saxitoxin average recovery rates are 85.0-
117.4%, and in a few days the reproducibility in the daytime (RSD) of reproducibility sum is below 13.2%.This shows the QuEChERS after optimization
Method has good accuracy and precision.
The recovery of standard addition and relative standard deviation (RSDs) (n=3) of 7 three kinds of various concentration saxitoxins of table
The concentration of fat-soluble saxitoxin in shellfish samples in greater coasting area is detected with the method established, as a result such as table 8
It is shown.SPX1, AZA3 and h-YTX are detected in shellfish samples, and SPX1, concentration are all contained in almost all of shellfish samples
Range is 0.14 μ g/kg-19.92 μ g/kg, and YTX, DTX1, DTX2, OA, AZA1 and AZA2 are below detection limit and European Union is maximum
Residue limits.
In 8 greater coasting area of table in shellfish samples fat-soluble saxitoxin concentration
The present invention establishes the UPLC-MS/MS quantitative detecting method of 9 kinds of fat-soluble saxitoxins in shellfish aquatic products：This method
Have many advantages, such as that quick, easy, efficient, the rate of recovery is high, organic solvent exposure is few, time saving.Before the QuEChERS sample of foundation
Reason technology can effectively remove complex matrices (as dissociated in conjunction with freezing grease removal technology and the high characterization of adsorption of graphene oxide
Fatty acid and pigment), clean-up effect is good.QuEChERS method after optimization, in conjunction with superelevation Liquid Chromatography-Tandem Mass Spectrometry
(UPLC-MS/MS) detection of fat-soluble saxitoxin in shellfish samples is applied successfully in technology, has preferable applicability.
Claims (7)
1. the QuEChERS method based on graphene establishes the UPLC-MS/MS detection method of fat-soluble saxitoxin, feature exists
In the detection method is to extract shellfish samples by QuEChERS method, assists QuEChERS method pair by cryogenic freezing technology
The clean-up effect of sample, then by being analyzed into UPLC-MS/MS；Wherein purifying adsorbent uses graphene.
2. the UPLC-MS/MS that the QuEChERS method according to claim 1 based on graphene establishes fat-soluble saxitoxin
Detection method, which is characterized in that the detection method the specific steps are：
1) QuEChERS method pre-treatment step：The good shellfish samples of 1g ± 0.01g homogeneous are accurately weighed in 15mL centrifuge tube, are added
Enter 2mL extracting solution, be vortexed concussion 30s；0.50g anhydrous magnesium sulfate is added, be vortexed concussion 1min, and 2.3 × 103G is centrifuged 5min, turns
Supernatant is moved in another 15mL centrifuge tube；It repeats to extract according to above-mentioned steps primary；Merge twice supernatant be placed in refrigerator-
20 DEG C of freezing 2h；Nitrogen at 40 DEG C of filtrate obtained is blown to close dry, addition 1mL by funnel of the rapid mistake equipped with absorbent cotton after taking-up
Extracting solution redissolves；Then 50.0mg graphene oxide and 100.0mg anhydrous magnesium sulfate is added, be vortexed concussion 5min, and 1.0 × 104g
It is centrifuged 5min, supernatant is analyzed after 0.22 μm of nylon leaching film filters into UPLC-MS/MS；
2) preparation of standard solution：According to the solubility and property of each toxin standard items, suitable toxin is drawn with pipettor
Standard items list mark, with methanol constant volume, so that the concentration of h-YTX, YTX are 400ng/mL, the concentration of OA, DTX1, DTX2, SPX are
200ng/mL；The concentration of AZA1, AZA2, AZA3 are the standard working solution of 50ng/mL, freezen protective under the conditions of -20 DEG C.
3. the UPLC- that the QuEChERS method according to claim 1 or 2 based on graphene establishes fat-soluble saxitoxin
MS/MS detection method, which is characterized in that mobile phase selective flow phase A：0.15% ammonium hydroxide and Mobile phase B：Methanol, 9 kinds of toxin are excellent
Mass spectrometry parameters after change are：Ion source：ESI electric spray ion source, negative ions scan simultaneously；Detection mode：MRM reacts prison more
It surveys；Capillary voltage：ESI+：3.5kv；ESI-：3.0kv；Ion source temperature：145℃；Desolventizing temperature：450℃；Spraying electricity
Pressure：4000V；Taper hole throughput：50L/h；Desolventizing gas flow：750L/h.
4. the UPLC-MS/MS that the QuEChERS method according to claim 1 based on graphene establishes fat-soluble saxitoxin
Detection method, which is characterized in that UPLC-MS/MS chromatographic condition is：Chromatographic column：Waters XBridge C18 chromatographic column 100mm
× 2.1mm, 5.0 μm；Column temperature：40℃；Sample volume：10μL；Mobile phase：A：0.15% ammonium hydroxide；B：Methanol；Flow velocity：0.3mL/
min；Type of elution：Gradient elution.
5. the UPLC-MS/MS that the QuEChERS method according to claim 2 based on graphene establishes fat-soluble saxitoxin
Detection method, which is characterized in that step 1) extracting solution is methanol, ethyl alcohol and the isopropanol that volume ratio is 7: 2: 1.
6. the UPLC- that the QuEChERS method according to claim 1 or 2 based on graphene establishes fat-soluble saxitoxin
MS/MS detection method, which is characterized in that purifying adsorbent is graphene oxide, reduced graphene, amination graphene or carboxyl
Graphite alkene.
7. the UPLC-MS/MS that the QuEChERS method according to claim 6 based on graphene establishes fat-soluble saxitoxin
Detection method, which is characterized in that purifying adsorbent is graphene oxide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810304789.8A CN108872412B (en) | 2018-04-04 | 2018-04-04 | UPLC-MS/MS detection method for establishing fat-soluble shellfish toxin based on graphene QuEChERS method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810304789.8A CN108872412B (en) | 2018-04-04 | 2018-04-04 | UPLC-MS/MS detection method for establishing fat-soluble shellfish toxin based on graphene QuEChERS method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108872412A true CN108872412A (en) | 2018-11-23 |
| CN108872412B CN108872412B (en) | 2022-01-04 |
Family
ID=64326575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810304789.8A Active CN108872412B (en) | 2018-04-04 | 2018-04-04 | UPLC-MS/MS detection method for establishing fat-soluble shellfish toxin based on graphene QuEChERS method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108872412B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110174469A (en) * | 2019-04-24 | 2019-08-27 | 山东省海洋资源与环境研究院 | The detection method of tri- kinds of diarrhetic shellfish poisons of OA, DTX1 and DTX2 in seaweed |
| CN110907568A (en) * | 2019-12-17 | 2020-03-24 | 大连工业大学 | Method for extracting free fatty acid from solid or semisolid food by using matrix solid phase dispersion |
| CN111272901A (en) * | 2020-03-14 | 2020-06-12 | 广西壮族自治区水产科学研究院 | High-resolution mass spectrometry detection method for lipophilic toxins in shellfish |
| CN111435128A (en) * | 2020-04-03 | 2020-07-21 | 宁波市疾病预防控制中心 | Method for measuring 4 diester-type aconite alkaloids in blood |
| CN114019042A (en) * | 2021-10-29 | 2022-02-08 | 浙江工商大学 | Method and device for determining content of pelteobagrus fulvidraco protease inhibitor based on graphene |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102872810A (en) * | 2012-10-22 | 2013-01-16 | 武汉大学 | Solid-phase adsorption material and application thereof to QuEChERS method |
| CN102928528A (en) * | 2012-10-15 | 2013-02-13 | 中国水产科学研究院黄海水产研究所 | High performance liquid chromatography mass spectrometry detecting method of 16 fat soluble saxitoxins in shellfish meet |
| CN104090057A (en) * | 2014-07-10 | 2014-10-08 | 江苏省产品质量监督检验研究院 | Matrix solid-phase dispersing agent of graphene oxide mixed material and application of matrix solid-phase dispersing agent |
| US20150377845A1 (en) * | 2014-04-16 | 2015-12-31 | King Fahd University Of Petroleum And Minerals | Fully automated analytical method for determination of chloroethers in water and urine sample |
-
2018
- 2018-04-04 CN CN201810304789.8A patent/CN108872412B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102928528A (en) * | 2012-10-15 | 2013-02-13 | 中国水产科学研究院黄海水产研究所 | High performance liquid chromatography mass spectrometry detecting method of 16 fat soluble saxitoxins in shellfish meet |
| CN102872810A (en) * | 2012-10-22 | 2013-01-16 | 武汉大学 | Solid-phase adsorption material and application thereof to QuEChERS method |
| US20150377845A1 (en) * | 2014-04-16 | 2015-12-31 | King Fahd University Of Petroleum And Minerals | Fully automated analytical method for determination of chloroethers in water and urine sample |
| CN104090057A (en) * | 2014-07-10 | 2014-10-08 | 江苏省产品质量监督检验研究院 | Matrix solid-phase dispersing agent of graphene oxide mixed material and application of matrix solid-phase dispersing agent |
Non-Patent Citations (4)
| Title |
|---|
| ARJEN GERSSEN等: "Liquid chromatography–tandem mass spectrometry method for the detection of marine lipophilic toxins under alkaline conditions", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| ASHOK KUMAR SUNDRAMOORTHY等: "Applications of graphene in quality assurance and safety of food", 《TRENDS IN ANALYTICAL CHEMISTRY》 * |
| 刘琳琳等: "超高效液相色谱－串联质谱法测定织纹螺中腹泻性贝类毒素的实验研究", 《中国预防医学杂志》 * |
| 韩深等: "超高压液相色谱-高分辨质谱快速筛查和确证食用贝类中多种原多甲藻酸贝类毒素", 《食品科学》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110174469A (en) * | 2019-04-24 | 2019-08-27 | 山东省海洋资源与环境研究院 | The detection method of tri- kinds of diarrhetic shellfish poisons of OA, DTX1 and DTX2 in seaweed |
| CN110174469B (en) * | 2019-04-24 | 2022-04-08 | 山东省海洋资源与环境研究院 | Detection method of three diarrheic shellfish poisoning types OA, DTX1 and DTX2 in seaweed |
| CN110907568A (en) * | 2019-12-17 | 2020-03-24 | 大连工业大学 | Method for extracting free fatty acid from solid or semisolid food by using matrix solid phase dispersion |
| CN111272901A (en) * | 2020-03-14 | 2020-06-12 | 广西壮族自治区水产科学研究院 | High-resolution mass spectrometry detection method for lipophilic toxins in shellfish |
| CN111435128A (en) * | 2020-04-03 | 2020-07-21 | 宁波市疾病预防控制中心 | Method for measuring 4 diester-type aconite alkaloids in blood |
| CN114019042A (en) * | 2021-10-29 | 2022-02-08 | 浙江工商大学 | Method and device for determining content of pelteobagrus fulvidraco protease inhibitor based on graphene |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108872412B (en) | 2022-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108872412A (en) | QuEChERS method based on graphene establishes the UPLC-MS/MS detection method of fat-soluble saxitoxin | |
| Lajeunesse et al. | Detection and confirmation of saxitoxin analogues in freshwater benthic Lyngbya wollei algae collected in the St. Lawrence River (Canada) by liquid chromatography–tandem mass spectrometry | |
| Lara-Martín et al. | Multi-residue method for the analysis of synthetic surfactants and their degradation metabolites in aquatic systems by liquid chromatography–time-of-flight-mass spectrometry | |
| CN102928528B (en) | High performance liquid chromatography-mass spectrometry detecting method of 16 fat soluble saxitoxins in shellfish meet | |
| CN102520092B (en) | Rapid detection method for discriminating adulteration of rice syrup in honey | |
| CN110146632B (en) | Liquid chromatography-mass spectrometry detection method for various marine biotoxins in aquatic products | |
| Chen et al. | Simultaneous screening for lipophilic and hydrophilic toxins in marine harmful algae using a serially coupled reversed-phase and hydrophilic interaction liquid chromatography separation system with high-resolution mass spectrometry | |
| CN105044249B (en) | Rapid-detection pretreatment kit for 28 polychlorinated biphenyls in aquatic product and application of kit | |
| CN105067697B (en) | A kind of phosphatide classification and Detection and quantitative approach based on stable isotope labeling | |
| CN107655991B (en) | Method for measuring 6 kinds of phthalate in soil | |
| CN106153801A (en) | A kind of method of seven kinds of mycotoxins in Chinese liquor supplementary material of detection simultaneously | |
| Chen et al. | Vortex-homogenized matrix solid-phase dispersion for the extraction of short chain chlorinated paraffins from indoor dust samples | |
| Zhang et al. | Polymeric ion exchange material based dispersive micro solid-phase extraction of lipophilic marine toxins in seawater followed by the Q Exactive mass spectrometer analysis using a scheduled high resolution parallel reaction monitoring | |
| CN112379015A (en) | Liquid chromatography-tandem mass spectrometry detection method for paralytic shellfish toxins in shellfish | |
| CN106706829A (en) | Method for measuring diarrhetic shellfish poisons in shellfishes by use of immunoaffinity purification-liquid chromatography-tandem mass spectrometry | |
| CN110174470B (en) | High-flux detection method for marine biotoxin in aquatic product | |
| Oliver-Rodríguez et al. | Multi-residue method for the analysis of commonly used commercial surfactants, homologues and ethoxymers, in marine sediments by liquid chromatography-electrospray mass spectrometry | |
| CN112198265A (en) | Pretreatment method, detection method and kit for simultaneously detecting multiple steroid hormones in blood sample | |
| Zhao-Yong et al. | Rapid screening and identification of paralytic shellfish poisoning toxins in red tide algae using hydrophilic interaction liquid chromatography-high resolution mass spectrometry with an accurate-mass database | |
| RU2425380C1 (en) | Method of quantitative analysis of urine dimethyl terephthalate by liquid chromatography | |
| CN107860858A (en) | A kind of method for high-flux analysis of mycotoxin in plant medicine material | |
| CN102944635B (en) | Method for determining tris (2,3-dibromopropyl) phosphate content of water | |
| CN101126751B (en) | Gas phase chromatogram- ion trap tandem mass spectrometry method for detecting organochlorine pesticide | |
| CN109001333A (en) | The method that Liquid Chromatography-Tandem Mass Spectrometry measures 3 kinds of short and naked dinoflagellate toxins in shellfish | |
| CN111085006A (en) | Process for extracting organic pollutants in environment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |