CN108872412B

CN108872412B - UPLC-MS/MS detection method for establishing fat-soluble shellfish toxin based on graphene QuEChERS method

Info

Publication number: CN108872412B
Application number: CN201810304789.8A
Authority: CN
Inventors: 赵巧灵; 王萍亚; 黄朱梁; 戴意飞; 陈翔; 蒋玲波
Original assignee: Zhoushan Institute For Food And Drug Control
Current assignee: Zhoushan Institute For Food And Drug Control
Priority date: 2018-04-04
Filing date: 2018-04-04
Publication date: 2022-01-04
Anticipated expiration: 2038-04-04
Also published as: CN108872412A

Abstract

The invention relates to a UPLC-MS/MS detection method for establishing fat-soluble shellfish toxin based on a graphene QuEChERS method. The invention establishes a UPLC-MS/MS quantitative detection method of 9 fat-soluble shellfish toxins in shellfish aquatic products, which comprises the following steps: the method has the advantages of rapidness, simplicity, convenience, high efficiency, high recovery rate, less organic solvent contact, time saving and the like; the established QuEChERS sample pretreatment technology is combined with the freezing degreasing technology and the high adsorption characteristic of graphene oxide, so that complex matrixes (such as free fatty acid and pigment) can be effectively removed, a better detection recovery rate effect of fat-soluble shellfish toxin is obtained, the purification effect is good, the low-temperature freezing technology is introduced to assist the purification effect of the QuEChERS method on the samples, the matrix benefit can be obviously reduced, and the sensitivity of the method is improved; by combining the ultra-high liquid chromatography tandem mass spectrometry technology, the method successfully applies the detection of the fat-soluble shellfish toxin in the shellfish sample, can simultaneously detect 9 fat-soluble shellfish toxins, obviously increases 5 kinds compared with the existing standard method, and has better applicability.

Description

UPLC-MS/MS detection method for establishing fat-soluble shellfish toxin based on graphene QuEChERS method

Technical Field

The invention relates to a detection method of fat-soluble shellfish toxin, in particular to a UPLC-MS/MS multi-component quantitative detection method for establishing fat-soluble shellfish toxin based on a QuEChERS method of graphene oxide, which reduces matrix benefit and improves sensitivity of the method.

Background

The marine biotoxin (shellfish toxin) is a large-class small-molecule toxic chemical substance which is mainly produced by marine toxic microalgae or microorganisms, can be enriched in marine organisms, particularly bivalve shellfish and is harmful to other organisms including human beings. With respect to these biotoxins, researchers initially classified them into six major groups based primarily on the symptoms of toxicity caused: paralytic Shellfish Poison (PSP), Diarrhetic Shellfish Poison (DSP), Neurogenic Shellfish Poison (NSP), memory-deficit Shellfish poison (ASP), Cigera Fistul Poison (CFP), cyanobacterial toxin (CFP). However, with the progress of research, new biotoxin species are continuously discovered, and various toxins such as OA are often associated with the toxins AZA and PTX, but have different toxicity mechanisms, and the original classification method cannot meet the requirements of management and scientific research. Thus, in 2004, the bivalve soft biotoxin working group, which was commonly established by the food and agriculture organization, the world health organization, and the inter-government marine committees, classified shellfish toxins into eight major groups, which were, respectively, Saxitoxin group (STX), Domoic acid group (Domoic acid, DA), Okadaic acid group (OA), azaspirane group (AZA), gymnodinium brevitum group (brevitoxin, BTX), Saxitoxin group (petotoxins, PTX), patinopectoxin group (YTX), and Cyclic imine group toxins (CIs). In addition to this, hydranth toxins (P1 TX) and Ciguatoxin (CTX) are also being considered as shellfish toxins.

In the existing 8 kinds of shellfish toxins, the STX and DA toxin groups are relatively easy to dissolve in water, and OA, AZA, BTX, PTX, YTX, and CIs are polyether substances, which have thermal stability and are easily dissolved in nonpolar organic reagents such as methanol and ether, and thus are collectively called as fat-soluble shellfish toxins (LPs). The serious hazard of the shellfish poison has attracted much attention in a plurality of countries, and developed countries such as Europe, America and the like have successively made shellfish poison monitoring national plans. The traditional monitoring method is mainly used for regularly monitoring the content of shellfish toxins and the types and the quantity of toxigenic algae of bivalve shellfish in related sea areas so as to evaluate the risk of the shellfish toxins, and makes positive contribution to protecting the safety of consumers and ensuring the healthy development of shellfish industry. However, the traditional means needs to collect a large amount of shellfish and algae samples to make effective early warning on shellfish toxins, which not only consumes a large amount of manpower and material resources, but also has weak timeliness. In 2004, MacKenzie et al reported for the first time a Solid phase adsorption toxin tracking technique (SPATT), which is mainly to adsorb target toxins by using specific adsorption materials, then to elute, concentrate and purify in a laboratory and then to detect by LC-MS. Compared with the traditional method, the SPATT technology not only greatly reduces the workload of sampling and analyzing, but also can monitor the change of shellfish toxins in different water layers of a target sea area, and has early warning effect on shellfish toxins by combining the change conditions of shellfish toxins and toxic algae, thereby having great development prospect. The existing detection technology is mainly a mouse biological method, has the problems of animal welfare, high detection limit, incapability of clearly distinguishing shellfish poison components and the like, and although the mass spectrometry detection technology is also used for detecting shellfish toxins, the detection type is single or a pretreatment method needs to be further improved.

Disclosure of Invention

The invention aims to solve the defect that the existing mass spectrometric detection method for shellfish toxins has single detection type, and provides a UPLC-MS/MS multi-component quantitative detection method for establishing fat-soluble shellfish toxins by a QuEChERS method based on graphene oxide, which reduces matrix benefits and improves the sensitivity of the method.

In order to achieve the purpose, the invention adopts the following technical scheme:

establishing a UPLC-MS/MS detection method of fat-soluble shellfish toxin based on a graphene QuEChERS method, wherein the detection method comprises the steps of extracting shellfish samples by the QuEChERS method, assisting the purification effect of the QuEChERS method on the samples by a low-temperature freezing technology, and then carrying out UPLC-MS/MS analysis; wherein the purification adsorbent uses graphene.

In the technical scheme, (1) graphene oxide is used as a purification adsorbent of the QuEChERS method for the first time, and a good detection recovery rate effect of fat-soluble shellfish toxin can be obtained. (2) The low-temperature freezing technology is introduced to assist the purification effect of the QuEChERS method on the sample, so that the matrix benefit can be obviously reduced, and the sensitivity of the method can be improved. (3) The UPLC-MS/MS established by the method can simultaneously detect 9 fat-soluble shellfish toxins, and 5 fat-soluble shellfish toxins are obviously increased compared with the existing standard method.

Preferably, the detection method comprises the following specific steps:

1) the QuEChERS method pretreatment step: accurately weighing 1g +/-0.01 g of homogenized shellfish sample into a 15mL centrifuge tube, adding 2mL of extracting solution, and performing vortex oscillation for 30 s; adding 0.50g anhydrous magnesium sulfate, vortex and shake for 1min, 2.3 × 10³g, centrifuging for 5min, and transferring the supernatant into another 15mL centrifuge tube; repeating the extraction according to the above steps; mixing the two supernatants, and freezing at-20 deg.C for 2 hr; taking out, quickly passing through a funnel filled with absorbent cotton, blowing the obtained filtrate to be nearly dry at 40 ℃ by nitrogen, and adding 1mL of extracting solution for redissolving; then 50.0mg of graphene oxide and 100.0mg of anhydrous magnesium sulfate are added, vortex and shake for 5min, 1.0 × 10⁴g, centrifuging for 5min, filtering the supernatant through a 0.22 mu m nylon filter membrane, and performing UPLC-MS/MS analysis;

2) preparation of a standard solution: according to the solubility and the property of each toxin standard, sucking a proper amount of toxin standard single standard by a pipette, and fixing the volume by methanol to ensure that the concentrations of h-YTX and YTX are 400ng/mL and the concentrations of OA, DTX1, DTX2 and SPX are 200 ng/mL; AZA1, AZA2, AZA3 were frozen at-20 ℃ in standard working solutions with a concentration of 50 ng/mL.

Preferably, the mobile phase is selected from mobile phase a: 0.15% ammonia and mobile phase B: the mass spectrum parameters after methanol and 9 kinds of toxin optimization are as follows: an ion source: ESI electrospray ion source, positive and negative ions scan simultaneously; the detection mode is as follows: monitoring MRM multiple reactions; capillary voltage: ESI⁺：3.5kv；ESI^-: 3.0 kv; ion source temperature: 145 ℃; desolventizing temperature: at 450 ℃; spraying voltage: 4000V; taper hole gas flow: 50L/h; desolventizing agent gas flow: 750L/h.

Preferably, the UPLC-MS/MS chromatographic conditions are as follows: a chromatographic column: waters Xbridge C18 column 100mm × 2.1mm, 5.0 μm; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; mobile phase: a: 0.15% ammonia; b: methanol; flow rate: 0.3 mL/min; and (3) an elution mode: gradient elution.

Preferably, the volume ratio of the extracting solution in the step 1) is 7: 2: 1 methanol/ethanol/isopropanol.

Preferably, the purification adsorbent is graphene oxide, reduced graphene, aminated graphene or carboxylated graphene.

The invention has the beneficial effects that:

the invention establishes a UPLC-MS/MS quantitative detection method of 9 fat-soluble shellfish toxins in shellfish aquatic products, which comprises the following steps: the method has the advantages of rapidness, simplicity, convenience, high efficiency, high recovery rate, less organic solvent contact, time saving and the like;

(1) the established QuEChERS sample pretreatment technology is combined with the freezing degreasing technology and the high adsorption characteristic of graphene oxide, so that complex matrixes (such as free fatty acid and pigment) can be effectively removed, a better detection recovery rate effect of fat-soluble shellfish toxin is obtained, the purification effect is good, the low-temperature freezing technology is introduced to assist the purification effect of the QuEChERS method on the samples, the matrix benefit can be obviously reduced, and the sensitivity of the method is improved;

(2) the optimized QuEChERS method combines the ultra-high liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technology, successfully applies the detection of the fat-soluble shellfish toxins in the shellfish samples, can simultaneously detect 9 fat-soluble shellfish toxins, obviously increases 5 fat-soluble shellfish toxins compared with the existing standard method, and has better applicability.

Drawings

FIG. 1 is the recovery of fat soluble shellfish poison with the present invention C18, PSA, GCB and GO as adsorbents.

Fig. 2 shows the recovery rates of fat-soluble shellfish poison when the graphene oxide, the reduced graphene, the aminated graphene and the carboxylated graphene are used as the adsorbent.

Detailed Description

The invention is further explained below with reference to specific embodiments and the attached drawings:

laboratory apparatus

Waters ultra-high liquid phase system (ACQUITYUPLC) and triple quadrupole tandem mass spectrometer (XEVO-TQ), electrospray ion source (ESI) and atmospheric pressure chemical ionization source (APCI) (Waters, USA);

a chromatographic column: waters Xbridge C18 column (5 μm, 100mmX2.1mm i.d.)₃column(1.8μm，，100mm×2.1mm i.d.，)Atlantics HILIC Silica column(Waters，150mm×2.1mm i.d.，5μm)(Waters，MA，USA)；

A water purifier: ELGA PURELA Ultra + Hitech RoDI plus pure water preparation instrument (ELGA, UK);

homogenizing: PoMron (PT2000, Switzerland);

freezing a centrifuge: type 3K-18(Sigma, Germany);

a vortex oscillator: SCILOGXE MX-S (SCILOGEX, USA);

tissue masher: IKA T-18(IKA, Germany);

analytical balance: ELECTRONIC BALANCE (SHIMADZU, Japan);

nitrogen blowing instrument: N-EVAP-24 (organic Association, USA);

ultrasonic cleaner: shanghai Kedao ultrasonic instruments, Inc.;

experimental reagent

Acetonitrile: HPLC grade, purity > 99.9%, available from Thermo Fisher Scientific, USA;

methanol: HPLC grade, purity > 99.9%, available from teiia company.in USA;

isopropyl alcohol: HPLC grade, purity > 99.8%, available from teiia company.in USA;

acetone: HPLC grade, purity > 99.5%, available from teiia company.in USA;

n-hexane: HPLC grade, purity > 99.5%, purchased from Honeywell, USA;

ethanol: analytically pure, with purity more than 99.7%, purchased from China pharmaceutical group headquarters;

ammonia water: the purity is 25-28%, and the product is purchased from China pharmaceutical group general company;

ultrapure water: the conductivity is more than or equal to 18.2 MOmega;

solid phase extraction column: OASIS HLB is purchased from Waters, USA;

Cleanert C₁₈PSA, GCB, available from Agela Technologies, USA;

graphene adsorption material: purchased from Nanoinnova technologies company (Madrid, Spain);

anhydrous magnesium sulfate (MgSO)₄) Purchased from the pharmaceutical group headquarters, china;

the 9 standard toxins (OA, DTX1, DTX2, YTX, h-YTX, SPX, AZA1, AZA2, AZA3) were purchased from National Research Council, Halifax, NS, Canada, National institute of Marine biology.

The detection method comprises the following specific steps:

1) the QuEChERS method pretreatment step: accurately weighing 1g +/-0.01 g of homogenized shellfish sample into a 15mL centrifuge tube, adding 2mL of extracting solution, carrying out vortex oscillation for 30s, wherein the volume ratio of methanol/ethanol/isopropanol is 7: 2: 1; adding 0.50g anhydrous magnesium sulfate, vortex and shake for 1min, 2.3 × 10³g, centrifuging for 5min, and transferring the supernatant into another 15mL centrifuge tube; repeating the extraction according to the above steps; mixing the two supernatants, and freezing at-20 deg.C for 2 hr; taking out, quickly passing through a funnel filled with absorbent cotton, blowing the obtained filtrate to be nearly dry at 40 ℃ by nitrogen, and adding 1mL of extracting solution for redissolving; then 50.0mg of graphene oxide and 100.0mg of anhydrous magnesium sulfate are added, vortex and shake for 5min, 1.0 × 10⁴g, centrifuging for 5min, filtering the supernatant through a 0.22 mu m nylon filter membrane, and performing UPLC-MS/MS analysis;

The purification adsorbent is graphene oxide, reduced graphene, aminated graphene or carboxylated graphene.

Shellfish samples are collected in the harbor sea area of Ningbo city in Zhejiang province. Cleaning the collected shellfish sample with clear water, taking out the whole fresh soft tissue, mashing in a tissue mashing machine, and preserving the obtained sample at-80 ℃.

Suitable liquid chromatography conditions are essential to improve chromatographic selectivity and reduce signal interference during mass spectrometry detection. Based on previous reports, the basic mobile phase will generally provide a more perfect selectivity, accuracy and lower limit of quantitation (LOQ) for the detection of fat-soluble shellfish toxins. In the invention, 0.15% ammonia water (mobile phase A) and methanol (mobile phase B) are selected as basic mobile phases, and the mass spectrum parameters of 9 optimized toxins are as follows:

an ion source: electrospray ion source (ESI), positive and negative ions are scanned simultaneously;

the detection mode is as follows: multiple Reaction Monitoring (MRM);

capillary voltage: ESI⁺：3.5kv；ESI^-：3.0kv；

Ion source temperature: 145 ℃;

desolventizing temperature: at 450 ℃;

spraying voltage: 4000V;

taper hole gas flow: 50L/h;

desolventizing agent gas flow: 750L/h

Mass spectrum multiple reaction monitoring conditions such as the taper hole voltage and the collision energy are shown in the table 1.

TABLE 1 Mass Spectrometry Multiple Reaction Monitoring (MRM) conditions for fat-soluble shellfish toxins

Establishment of fat-soluble shellfish poison UPLC-MS/MS chromatographic condition

A chromatographic column: waters Xbridge C18 chromatography column (100 mm. times.2.1 mm, 5.0 μm);

column temperature: 40 ℃;

sample introduction amount: 10 mu L of the solution;

mobile phase: a: 0.15% ammonia; b: methanol;

flow rate: 0.3 mL/min;

and (3) an elution mode: gradient elution;

the gradient elution procedure is shown in table 2.

TABLE 2 gradient elution of target shellfish toxin

Time (min)	Flow rate (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 optimized quantitative detection methodological verification of QuEChERS combined with an ultra-high liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for detecting the shellfish toxin comprises the following parameters: linearity, matrix effect, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). Because the shellfish toxin has no proper internal standard, an external standard method is selected to be matched with a matrix matching curve for quantification. The matrix matching curve is obtained by adding a shellfish toxin mixing standard product with known concentration through a blank matrix according to gradient concentration, wherein the blank matrix liquid is obtained by purifying optimized QuEChERS of the shellfish sample crude extract. All experiments were performed in triplicate. Linearity of the standard curve isBy a correlation coefficient (r)²) Determination of day-to-day reproducibility (Intra-day) and day-to-day reproducibility (Inter-day) as well as precision was judged by determination of recovery and Relative Standard Deviation (RSD) using a matrix spiking experiment. Detecting the detection Limit (LOD) and the quantification Limit (LOQ) by adding a standard solution to a substrate, and taking the signal-to-noise ratio (S/N) as the quantification Limit (LOQ) when the S/N is 10: 1; the dilution was continued until the signal to noise ratio (S/N) was 3: 1, which was taken as the limit of detection (LOD) of the method.

Optimization of extraction solvent

The complex matrix components in the shellfish extract are incompatible with the UPLC-ESI-MS/MS system. Therefore, in order to obtain higher mass spectrum signals, better recovery rate and higher sample preparation efficiency, the existing QuEChERS method is improved to have higher extraction efficiency and relatively simple operation process. Typically, the target analyte in the sample is extracted using a suitable solvent, which is the initial step of the queechers method. Therefore, methanol, ethanol, isopropanol, methanol: ethanol: isopropanol (7: 2: 1, v/v/v), methanol: ethanol: isopropanol (2: 7: 1, v/v/v), methanol: ethanol: isopropanol (4.5: 1, v/v/v) were respectively selected as extraction solvents, and the optimal extraction reagent was obtained by the recovery rate of the extracted shellfish toxin. The result shows that for most fat-soluble marine shellfish toxins, when methanol, ethanol and isopropanol (7: 2: 1, v/v/v) are selected as extracting agents, the recovery rates of the 9 shellfish toxins are all about 100%, the fluctuation is stable, and the optimal recovery rate is obtained.

Selection study of adsorbents

The matrix effect generally causes the enhancement or inhibition of mass spectrum signals of fat-soluble shellfish poison when in LC-MS/MS analysis. Therefore, the shellfish sample is immediately frozen after being preliminarily extracted, is placed in a lead box for 2 hours at the temperature of below 20 ℃ below zero, is adhered to the wall by paying attention to completely freeze the shellfish sample, and is quickly filtered to remove most of lipid in the shellfish sample. However, the color of the crude extract did not change significantly, indicating that the pigment in the crude extract from the shellfish sample was not removed by the freezing step. Typically, PSA, C18 and GCB are used for free fatty acid and pigment removal. Thus, their ability to remove interfering components was evaluated by comparing different types of adsorbents, including C18, PSA, GCB, and GO. The results show that the extract after clarification with GCB and GO tends to be more transparent than the clarification effect of PSA and C18. In addition, it was found that the matrix enhancement effect was significant in the crude extract purified with C18 and PSA, which is consistent with the tendency of YTXs and AZAs to signal enhancement by the matrix enhancement effect in LC-MS/MS analysis reported in the literature. Furthermore, although the pigment removal effect was difficult to distinguish between GCB and GO when using dispersed solid phase extraction (d-SPE) purification, the GO adsorbent showed better pigment removal capacity because in the linear range, GO provided satisfactory recovery between 82.3% and 111.7%, with a Relative Standard Deviation (RSD) below 10.0%, with the results shown in table 3.

TABLE 3 recovery of fat-soluble shellfish toxin and RSD with C18, PSA, GCB and GO as adsorbents

However, the recovery rate was lower during the purification due to the high retention of YTX and h-YTX by GCB adsorbent, 50.4% and 58.6% for h-YTX and YTX, respectively. The results show that GO serving as an adsorbent of the QuECHERS method has the best capability of removing impurities in the extracting solution, and obtains better recovery rate, wherein the recovery rate ranges from 88.6% to 108.3%. As shown in fig. 1.

Comparative study of graphene in different forms

The impurity removal capacity of four kinds of graphene, namely oxidized graphene, reduced graphene, aminated graphene and carboxylated graphene, when the four kinds of graphene are used as the QuECHERS method adsorbent is investigated, and the recovery rate and the Relative Standard Deviation (RSD) are used as evaluation indexes for comparison. The results are shown in table 4 and fig. 2. Among the four kinds of graphene, the graphene oxide is most stable to the adsorption of target toxins; the retention of the reduced graphene and the aminated graphene on h-YTX and YTX shellfish toxins is high, so that the recovery rate is too low; compared with graphite oxide, the overall recovery rate of the carboxylated graphene is lower than that of the graphene oxide. Thus, graphene oxide was selected as the purification adsorbent.

Table 4 recovery rates of fat-soluble shellfish toxins using graphene oxide, reduced graphene, aminated graphene, and carboxylated graphene as adsorbents

Optimization of mass spectrometry conditions

In order to provide better signal response during mass spectrometry quantification, each fat-soluble shellfish toxin standard is directly injected independently, and positive and negative ions are scanned simultaneously in a multi-reaction monitoring mode (MRM) to find that AZAs and SPX shellfish toxins are suitable for a positive ion mode, and YTXs, DTXs and OA shellfish toxins are suitable for a negative ion mode. Therefore, when 9 shellfish toxins are detected, the scanning is required to be continuously switched between the parent ions and the child ions with the highest response, and the ions with higher response values in the ion pairs are used as quantitative ions. For each analyte, parameters of tandem mass spectrometry (MS/MS) were optimized, including the cone-hole voltage, collision voltage, quantitative ions and qualitative ions, as shown in table 1.

Optimization of chromatographic conditions

Proper chromatographic separation conditions play a crucial role in improving resolution and reducing signal interference during mass spectrometry detection. In general, alkaline mobile phases may provide better sensitivity, accuracy, and lower quantitation and detection limits. In the present invention, the mobile phases are 0.15% ammonia (mobile phase a) and methanol (mobile phase B), and optimization by gradient elution provides sufficient resolution for separation of the fat-soluble shellfish poison.

Although the QuEChERS method can effectively remove matrix components and thus achieve high accuracy, the influence of matrix effects in the sample cannot be completely avoided in the routine LC-MS/MS analysis. The presence of interfering components in the matrix can have a matrix enhancing or matrix inhibiting effect on the target analyte, causing either quantitative duplication or quantitative deletion of the target species due to the high sensitivity of the ion source ESI in tracking the target species. Therefore, in the invention, in order to effectively avoid inaccurate quantification caused by the matrix effect, an external standard method and a matrix matching curve are used for evaluating the matrix effect. Meanwhile, the matrix effect is different due to different matrixes and shellfish toxins, so that in order to evaluate the stability of the QuEChERS method, two different matrix matching curves of clam and common mussel samples are used, and the slope of the matrix matching curves shows (table 5), the QuEChERS method based on GO and freezing degreasing steps can obviously eliminate main matrix components, and the linearity and the signal stability of the method are greatly improved. Meanwhile, the result shows that the matrix matching curve using the specific shellfish extract can be used for the determination of other shellfish samples.

TABLE 5 evaluation Table of matrix Effect in Meretrix Linnaeus and Mytilus edulis

Linearity, LOD (detection limit) and LOQ (quantitative limit)

And (3) processing the sample according to an optimized sample pre-method to obtain 5 series blank adding patterns with the target object content, and then carrying out UPLC-MS/MS detection. A standard curve is plotted with the response area y of the target analyte as the ordinate and the target analyte addition concentration X (. mu.g/kg) as the abscissa. As can be seen from table 6, in the constructed matrix matching curves, 9 fat-soluble shellfish toxins: h-YTX and YTX are 6.0-80.0 μ g/kg; DTX1, DTX2, OA and SPX are 3.0-40.0 mu g/kg; AZA1, AZA2, AZA3 were in a linear relationship of 0.75-10.0. mu.g/kg. The results show that the correlation coefficient (r)²) The range is 0.9827-0.9997, which shows that the experiment obtains satisfactory linearity, and the optimized method has enough applicability and can meet the analysis requirement of quantitative detection of fat-soluble shellfish toxins. Table 6 shows that the method has a limit of detection (LOD) of 0.10 to 1.47. mu.g/kg and a limit of quantitation (LOQ) of 0.32 to 4.92. mu.g/kg, which is much lower than the Maximum Residual Limit (MRL) of the European Union. These data show that the process is excellentAnd (4) sensitivity.

TABLE 6 Linear Range, correlation coefficient (r) of fat-soluble shellfish toxin²) Limit of detection (LOD) and limit of quantification (LOQ)

Shellfish poison	Linear range/(μ g/kg)	Coefficient of correlation r²	Detection limit (mug/kg)	Limit of quantitation (ug/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

Recovery and precision

Shellfish is used as a sample, 3 horizontal matrix labeling experiments are carried out, the experiments are respectively repeated for 3 times, and the recovery rate and the precision are tested according to the sample pretreatment method in section 2.2.2. As can be seen from Table 7, the average recovery rates of the 9 fat-soluble shellfish toxins were 85.0-117.4%, and the daily and daytime Reproductions (RSD) were less than 13.2%. This indicates that the optimized QuEChERS method has good accuracy and precision.

TABLE 7 normalized recovery and Relative Standard Deviation (RSDs) (n ═ 3) for three different concentrations of shellfish poison

The established method was used to determine the concentration of fat-soluble shellfish toxins in shellfish samples in the offshore region, and the results are shown in table 8. SPX1, AZA3 and h-YTX were detected in shellfish samples, almost all shellfish samples contained SPX1 at a concentration range of 0.14. mu.g/kg-19.92. mu.g/kg, and YTX, DTX1, DTX2, OA, AZA1 and AZA2 were below the detection limit and the EU maximum residual limit.

TABLE 8 concentration of fat-soluble shellfish toxin in shellfish samples in the offshore region

The invention establishes a UPLC-MS/MS quantitative detection method of 9 fat-soluble shellfish toxins in shellfish aquatic products, which comprises the following steps: the method has the advantages of rapidness, simplicity, convenience, high efficiency, high recovery rate, less organic solvent contact, time saving and the like. The established QuEChERS sample pretreatment technology is combined with a freezing degreasing technology and the high adsorption characteristic of graphene oxide, so that complex matrixes (such as free fatty acid and pigment) can be effectively removed, and the purification effect is good. The optimized QuEChERS method is combined with an ultra-high liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technology, the detection of fat-soluble shellfish toxins in shellfish samples is successfully applied, and the method has good applicability.

Claims

1. The method is characterized in that a shellfish sample is extracted by a QuEChERS method, the sample is purified by a low-temperature freezing technology assisted QuEChERS method, and then UPLC-MS/MS analysis is carried out;

the detection method comprises the following specific steps:

1) the QuEChERS method pretreatment step: accurately weighing 1g +/-0.01 g of homogenized shellfish sample in a 15mL centrifuge tube, and adding 2mL of extracting solution, wherein the volume ratio of the extracting solution is 7: 2: 1 of methanol, ethanol and isopropanol; vortex oscillation is carried out for 30 s; adding 0.50g anhydrous magnesium sulfate, vortex and shake for 1min, 2.3 × 10³g, centrifuging for 5min, and transferring the supernatant into another 15mL centrifuge tube; repeating the extraction according to the above steps; mixing the two supernatants, and freezing at-20 deg.C for 2 hr; taking out, quickly passing through a funnel filled with absorbent cotton, blowing the obtained filtrate to be nearly dry at 40 ℃ by nitrogen, and adding 1mL of extracting solution for redissolving; then 50.0mg of graphene oxide and 100.0mg of anhydrous magnesium sulfate are added, vortex and shake for 5min, 1.0 × 10⁴g, centrifuging for 5min, filtering the supernatant through a 0.22 mu m nylon filter membrane, and performing UPLC-MS/MS analysis;

the UPLC-MS/MS chromatographic conditions are as follows: a chromatographic column: waters Xbridge C18 column 100mm × 2.1mm, 5.0 μm; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; mobile phase: a: 0.15% ammonia; b: methanol; flow rate: 0.3 mL/min; and (3) an elution mode: gradient elution; the gradient elution procedure was:

the mass spectrum conditions are as follows: mobile phase selection mobile phase a: 0.15% ammonia and mobile phase B: the mass spectrum parameters after methanol and 9 kinds of toxin optimization are as follows: an ion source: ESI electrospray ion source, positive and negative ions scan simultaneously; the detection mode is as follows: monitoring MRM multiple reactions; capillary voltage: ESI⁺：3.5kv；ESI^-: 3.0 kv; ion source temperature: 145 ℃; desolventizing temperature: at 450 ℃; spraying voltage: 4000V; taper hole gas flow: 50L/h; desolventizing agent gas flow: 750L/h; the mass spectrum multiple reaction monitoring conditions of the 9 fat-soluble shellfish toxins are as follows: