CN103983495A - Tetrodotoxin positive quality control sample as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a method for preparing a tetrodotoxin positive quality control sample. The method comprises the steps of selecting muscular tissues from fresh fugu rubripes, homogenizing, and measuring the content of tetrodotoxin in the muscular tissues to verify whether the muscular tissues contain the tetrodotoxin; if the muscular tissue is a positive sample, diluting the positive sample containing toxin with a negative sample and a dispersant, mixing uniformly, continuously measuring the content of the toxin in the sample, packaging the sample after the sample meets requirements, preserving at an appropriate temperature, and performing uniformity verification and stability analysis on the sample; if the muscular tissue is a negative sample, adding a tetrodotoxin standard solution into the negative samples, adding a dispersant, mixing uniformly, measuring the content of the tetrodotoxin in the sample, packaging the sample after the sample meets requirements, preserving at an appropriate temperature, and performing uniformity verification and stability analysis on the sample. The quality control sample is simple to operate, and has enough stability and uniformity and long preservation time, so that the quality control sample can be used for laboratory quality control.

Description

A kind of fugutoxin positive quality control sample and its preparation method and application

Technical field

The present invention relates to a kind of fugutoxin (Tetrodotoxin, TTX) positive quality control sample and preparation method thereof, belong to food safety detection technical field.

Background technology

Fugu (comprising Fugu rubripes (Fugu rubripes) and fugu obscurus (Fugu obscurus) etc.) is to propagate on a large scale at present Puffer class fingerling artificially, albumen in the flesh of fish and lipid content are abundant, and detoxification is precious delicious food after processing.In order to monitor Various Seasonal river Puffer fish toxicity, fast, measure exactly TTX content in the Puffer fish tissue of river, distinguish poisonous river Puffer and nontoxic river Puffer, understand in real time Difference In Toxicity and the situation of change of common river Puffer fish, a kind of rapid and convenient of very necessary foundation, highly sensitive, workable TTX detection method, and prepare practical fugutoxin positive quality control sample, can in TTX testing process, detect with actual sample simultaneously, to weigh the reliability of fugutoxin testing result in the Puffer fish matrix of river, significant to open Puffer fish market, domestic and international river to improving the edible safety of river Puffer fish, also for measurement and the research work of the aspects such as food security, environmental monitoring, sanitary inspection quarantine provide technical support and magnitude tracing guarantee.

River Puffer flesh of fish delicious, nutritious, eat raw, cook, process all suitable, but owing to containing hypertoxic fugutoxin (Tetrodotoxin, TTX), can not become so far the food on popular dining table.River Puffer poison, it is the one severe toxicity nerve toxin that river Puffer body contains, also be present in newt, spot foot toad and gastropod body, it is one of the most malicious non-protein substance of finding of current occurring in nature, its toxicity is equivalent to 1250 times of poisonous drugs potassium cyanide, only need 0.48mg can cause people desperately, be collectively referred to as the large public hazards toxin in ocean three with saxitoxin, liguatoxin.Along with the development of fishery, the end that appears in the newspapers repeatly of river Puffer ichthyism event in recent years.Poisoning at the edible river Puffer fish of Japan, China's Mainland, Taiwan Province and some countries in Southeast Asia and gastropod happens occasionally, there is report in poisonings such as China especially edible small-sized gastropod basket whelk more, and cause many people dead and become serious public health problem (P.A.Hwang et al, 2007).European Union's regulation " Regulation853/2004/EC " and " Regulation854/2004/EC " specify that poisonous river Puffer fish and similar kind forbid selling at EU market.

Positive quality control sample is a kind of same or analogous with sample to be measured, but confirms and have the material of specifying measurement uncertainty standard value with traceability, in the characteristic aspect storage value and transmission value with material measure.Thereby positive quality control sample is for ensureing reliability and the validity of testing result, ensure that examination criteria implements effectively and have vital role between laboratory.

At present existing fugutoxin standard substance, the for example fugutoxin standard model of National Bureau of Oceanography's the 3rd Research Institute (real is the purification standard items that do not contain HePuffer fish periplast), but the matrix standard model of fugutoxin there is not yet the report of research preparation

Because the character of fugutoxin is slightly soluble in water (toxin being contained in internal organs can be water-soluble), ethanol, concentrated acid, be soluble in diluted acid, be insoluble to any neutral organic solvent, comprise ether, methyl alcohol, ethanol, acetone, N, N '-dimethyl formamide, dimethyl sulfoxide, benzene, chloroform and acetonitrile etc.This physicochemical characteristics that is not soluble in any neutral organic solvent, and river Puffer fish organizes the existence of complicated matrix effect, the quantitative measurement that makes fugutoxin is a challenge of food inspection, ecology and medical research field always.For ensureing reliability and the validity of testing result, the urgently development of matrix TTX positive quality control sample at present.

Summary of the invention

Technical matters to be solved by this invention is: a kind of matrix TTX positive quality control sample is provided, and described quality-control sample has enough stability and homogeneity.

For solving the problems of the technologies described above, the technical solution used in the present invention is:

The invention provides a kind of preparation method of fugutoxin positive quality control sample, comprise that positive dilution and negative sample standard substance add two schemes, comprise the following steps: scarlet fin dongfang globe fish to get homogeneous after musculature, measure the wherein content of fugutoxin, whether checking contains fugutoxin, if positive, the positive that contains toxin is mixed with negative sample and spreading agent dilution, continue to measure the wherein content of toxin, reach after mark content requirement, by sample packing, under proper temperature, preserve, sample is carried out to homogeneity checking and stability analysis, if negative sample, adds fugutoxin standard solution in negative sample to, add spreading agent to mix, measure the wherein content of fugutoxin, reach after requirement, by sample packing, under proper temperature, preserve, sample is carried out to homogeneity checking and stability analysis.

The preparation method of fugutoxin positive quality control sample described above, specifically comprises the following steps:

(1) get scarlet fin dongfang globe fish and get musculature, homogenizer homogeneous, in homogenizing process, pressure is 25-35Mpa, homogenizing time adopts gradient Time Method, homogeneous three times altogether, each time is respectively 15-20min, 10-15min, 5-10min;

(2) sample good homogeneous is carried out to toxin determination, adopt conventional method to measure, according to whether containing toxin, preparation method is divided into positive dilution and negative sample standard is added two kinds;

1. positive dilution: the positive that contains toxin is mixed with negative sample and spreading agent dilution, spreading agent weight accounts for the 8-12% of positive and negative sample general assembly (TW), dilution mixes the stepwise dilution method that adopts, negative sample and spreading agent are divided into 3-5 part, after adding a negative sample and spreading agent, once, each homogenizing time is 3-5min to homogeneous;

2. negative sample standard is added: fugutoxin standard solution is added to after measured not containing in the scarlet fin dongfang globe fish musculature of fugutoxin, then average mark adds for 3-5 time spreading agent to mix, often add a spreading agent homogeneous once, each homogenizing time is 3-5min, adds the total amount of spreading agent to account for the 8-12% of negative sample total amount.

(3) content of toxins of sample in determination step (2);

(4) content of toxins reaches after mark content requirement, and by sample packing, proper temperature is preserved, and sample is carried out to homogeneity checking and stability analysis.

Preferably, in step (1), musculature before homogeneous, is heated to 40-50 DEG C by musculature in homogenizer, and when homogeneous, homogenizing temperature is controlled at 30-40 DEG C.Musculature in homogenizer before homogeneous heating and homogenizing process in keep uniform temperature, can be better by toxin stripping.

Described spreading agent is C ₁₈, the combination of a kind of in graphitized carbon, white carbon, zeyssatite or two kinds.Add spreading agent can be conducive to reduce sample substrate viscosity, be convenient to TTX toxin and be dispersed in matrix, make sample stable; Also with sample detection process in pretreatment process be consistent, in sample determination process, there is absorption impurity, purify the feature of extract.(between the carbon atom on action principle GCB surface, be all SP2 hydridization, have single electron to active ion, and there is hexagonal micromechanism, make it there is strong suction-operated for planar molecule or the molecule that contains plane aromatic rings, can remove and there is the impurity such as phenylalanine, tyrosine, tryptophane and the histidine of conjugated structure; GCB surface aggregate performance is hydrophobic nature, adsorbable nonpolar and low pole compound; Its subsurface exists some polarity sites to make it can serve as ion exchanger or adsorption of polar compounds, and adsorbable river Puffer fish flavor components glutamic acid, arginine, aspartic acid etc. under the extraction conditions of this experiment show as the absorption property of wide spectrum; C ₁₈also be a kind of nonpolar broad spectrum activity cleanser, effectively adsorbing and extracting non polar impurities; Zeyssatite has sieving actoion, in-depth filtration and reaches the effect of absorption impurity by intermolecular force (Van der Waals force), Zeta potential and ion exchange.)

Preferably, step (4) content of toxins reaches after mark content requirement, can continue sample freeze drying or oven dry, does powdering or wet-milling shape or pulpous state.

The mark content of toxin can be designated: 1～10 many of μ g/kg content series; Sample size is equivalent to respectively 1g sample, 2g sample, 3g sample, 4g sample, 5g sample etc., so that consistent with practical operation sample weighting amount.

Preferably, sample storage condition is that 4 DEG C, low temperature or ultralow temperature are preserved.

The present invention also provides a kind of preparation method who utilizes described fugutoxin positive quality control sample to obtain fugutoxin positive quality control sample.Does is what meaning (this sentence?)

The natural origin of quality-control sample mesostroma fugutoxin of the present invention (TTX) comprises the living organism such as toxic river Puffer fish or basket whelk of Nature creating, but be not limited to, these are several.

Fugutoxin in described quality-control sample (TTX) source is fugutoxin (TTX) standard substance (comprising natural purification and chemosynthesis).

The invention has the beneficial effects as follows:

Quality-control sample preparation of the present invention is simple, has enough stability and homogeneity; Holding time is long, can be used for laboratory quality control.The spreading agent adding has dispersion, stablizes, contributes to the functions such as the extraction and cleaning of pretreatment process in analyzing and testing program.

Embodiment

The present invention is further described in detail in conjunction with the embodiments, but is not limited to this.

(blank matrix standard addition method, fills a prescription as C for preparation and formulation Example 1 ₁₈with graphitized carbon powder, addition of C in this example ₁₈with graphitized carbon ratio be 1:1)

1) sampling Preliminary detection TTX are containing measuring the scarlet fin dongfang globe fish of propagating artificially, cutting off back epidermis gets muscle parts and avoids getting blood and internal organ, obtain 400g musculature, measure with analyzing muller (IKA A11B25) homogeneous post analysis, result is not for containing fugutoxin in this sample; 2) mark-on mixes and adds the standard solution that contains 20 μ g TTX, mixes 15min; 3) add spreading agent and stabilizing agent average mark to add spreading agent (C for 3-5 time ₁₈with the each 20g of graphitized carbon powder) mix, often add a spreading agent homogeneous once, each homogenizing time is 3-5min, add the total amount of spreading agent to account for 10% of negative sample total amount, fully mix rear with HPLC-MS/MS method mensuration TTX content, measurement result is in tolerance interval, then mensuration is carried out Homogeneity; 4) packing, frozen, stability inspection accurately take 1.10g sample and (be equivalent to 1.00g river Puffer flesh of fish sample) in 50ml polypropylene centrifuge tubes, seal, in-20 degrees Celsius of preservations, with 1 month, 2 months, half a year, 1 year for carrying out examine stability in interval.

(blank matrix standard addition method, fills a prescription as C for preparation and formulation Example 2 ₁₈with graphitized carbon powder, addition of C in this example ₁₈with graphitized carbon ratio be 2:1)

1) sampling Preliminary detection TTX are containing measuring the scarlet fin dongfang globe fish of propagating artificially, cutting off back epidermis gets muscle parts and avoids getting blood and internal organ, obtain 400g musculature, measure with analyzing muller (IKA A11B25) homogeneous post analysis, result is not for containing fugutoxin in this sample; 2) mark-on mixes and adds the standard solution that contains 20 μ g TTX, mixes 15min; 3) add spreading agent and stabilizing agent average mark to add spreading agent (C for 3-5 time ₁₈with graphitized carbon powder difference 26.7 and 13.3g) mix, often add a spreading agent homogeneous once, each homogenizing time is 3-5min, add the total amount of spreading agent to account for 10% of negative sample total amount, fully mix rear with HPLC-MS/MS method mensuration TTX content, measurement result is in tolerance interval, then mensuration is carried out Homogeneity; 4) packing, frozen, stability inspection accurately take 1.10g sample and (be equivalent to 1.00g river Puffer flesh of fish sample) in 50ml polypropylene centrifuge tubes, seal, in-20 degrees Celsius of preservations, with 1 month, 2 months, half a year, 1 year for carrying out examine stability in interval.

Formulation Example 3 (formula is single addition of C 18 powder)

1) sampling Preliminary detection TTX are containing measuring the scarlet fin dongfang globe fish of propagating artificially, cutting off back epidermis gets muscle parts and avoids getting blood and internal organ, obtain 400g musculature, measure with analyzing muller (IKA A11B25) homogeneous post analysis, result is not for containing fugutoxin in this sample; 2) mark-on mixes and adds the standard solution that contains 20 μ g TTX, mixes 15min; 3) add spreading agent and stabilizing agent average mark to add spreading agent (C for 3-5 time ₁₈40g) mix, often add a spreading agent homogeneous once, each homogenizing time is 3-5min, add the total amount of spreading agent to account for 10% of negative sample total amount, fully mix the rear HPLC-MS/MS of using method and measure TTX content, measurement result is in tolerance interval, then mensuration is carried out Homogeneity; 4) packing, frozen, stability inspection accurately take 1.10g sample and (be equivalent to 1.00g river Puffer flesh of fish sample) in 50ml polypropylene centrifuge tubes, seal, in-20 degrees Celsius of preservations, with 1 month, 2 months, half a year, 1 year for carrying out examine stability in interval.

Formulation Example 4

Additive formulations is C ₁₈powder, additive formulations is C ₁₈, graphitized carbon and zeyssatite ratio be 2:2; 1, but be not limited to this.

Use embodiment 1

Sample pretreatment

1) the freezing execution of river Puffer fish of living, peeling, gets its muscle parts and goes muscle fully to pulverize through homogeneous sampling machine.

2) take sample (1.00 ± 0.01) g in 50mL polypropylene centrifuge tube, accurately add 1% acetic acid methyl alcohol (1:99, v/v) solution 5mL, homogeneous 30s, centrifugal in 50 DEG C of ultrasonic extraction 25min.8000r/min*5min, shift supernatant to another clean centrifuge tube; Residue repeats to extract once with 4mL1% acetic acid methanol solution, merges supernatant in 4 DEG C of centrifugal 5min of 12000r/min, and gained supernatant is to be clean;

3) get 1.0mL supernatant in 2mL EP (Eppendorf) pipe, add 100mg ketjenblack EC (GCB) and 100mg C ₁₈the violent vortex mixed 30s of powder, the centrifugal 5min of 10000r/min, supernatant liquor is collected in plastics sample introduction bottle HPLC-MS/MS and measures after 0.22 μ m teflon-coextrusion nylon film filters.

Positive quality control sample is measured the fugutoxin positive quality control sample of getting 50mL polypropylene centrifuge tube and packing and (is equivalent to river Puffer flesh of fish matrix (1.00 ± 0.01) g), accurately add 1% acetic acid methyl alcohol (1:99, v/v) solution 5mL, homogeneous 30s, centrifugal in 50 DEG C of ultrasonic extraction 25min.8000r/min*5min, shift supernatant to another clean centrifuge tube; Residue repeats to extract once with 4mL1% acetic acid methanol solution, merges supernatant in 4 DEG C of centrifugal 5min of 12000r/min, and gained supernatant is to be clean; All the other are processed with 1.23)

Homogeneity and stability test

Homogeneity is investigated

Carry out homogeneity research by the rule of standard model work directive/guide (3) standard model definite value and statistical method (GB/T15000.3-2008/ISOGuide35:2006) 5.8, by 2 × N of total sample unit number N ^1/3randomly drawing sample.

Detect by " mensuration of fugutoxin in the Puffer fish of the fresh river of GB/T5009.206-2007 " and " high performance liquid chromatography/tandem mass spectrometry is measured TTX in the Puffer tissue of river ", each sample repeated test 2 times; Homogeneity between the bottle of employing method of analysis of variance (F-inspection) check sample.Sample homogeneity assay is in table 1.

Table 1 fugutoxin positive quality control sample homogeneity assay (μ g/kg)

According to the sample homogeneity method of inspection, once calculate

m＝13 N＝26

Population mean $\stackrel{=}{x}=72.85$

Sum of squares between groups $Q_1=\underset{i=1}{\overset{m}{\mathrm{\Σ}}}{n}_i{({\stackrel{\‾}{x}}_i-\stackrel{=}{x})}^2=0.85235$

Quadratic sum in group

$Q_2=\underset{i=1}{\overset{m_i}{\mathrm{\Σ}}}\underset{j=1}{\overset{n_i}{\mathrm{\Σ}}}{(x_j-{\stackrel{\‾}{x}}_i)}^2=0.79250$

v ₁＝m-1＝12

v ₂＝N-m＝13

The amount of taking statistics F

$F=\frac{\frac{Q_1}{v_1}}{\frac{Q_1}{v_2}}=\frac{0.07103}{0.06096}=1.17$

Look into subordinate list 1F distribution tables of critical values and obtain F _0.05(12,13)=2.60, F < F _0.05(12,13)

By comparing between-group variance and interclass variance, the two ratio is less than the critical value of statistical test, therefore the content distribution of TTX is uniform in fugutoxin positive quality control sample.

Study on the stability

In JJG1006-1994, specify:

" standard substance should, under the storage of regulation or service condition, carry out the stability test of characteristic value undetermined termly.“

" time interval of stability test can be by first close rear thin principle arrangement.Within the valid period, should there is the Monitoring Data in multiple time intervals.”

" selection is not less than valued methods precision and carries out stability test with the measuring method with enough sensitivity, and notes the consistent of operation and experiment condition.”

" investigate stability specimen in use and should from be distributed into the sample of minimum package unit, randomly draw, the sample number of extraction has enough representativenesses for overall sample.”

Stability to fugutoxin is monitored, in 6 months, respectively the Stability Determination to fugutoxin 4 times, get at random 3 fugutoxin samples at every turn, application t method of inspection carries out estimation of stability.

T method of inspection: in the time that the standard value of standard substance characteristic value is known, the stability of available t method of inspection evaluation criterion material, by formulae express is

$|{\stackrel{\&OverBar;}{X}}_i-\stackrel{\&OverBar;}{X}|\&le;t_a(n-1)\&CenterDot;\frac{s}{\sqrt{n}}$

In formula, for the measurement mean value of arbitrary standard substance STABILITY MONITORING; for the standard value of standard substance characteristic value; t _a(n-1) be t inspection critical value; S is the standard deviation of measuring method; N is for measuring number of times.

If with t inspection critical value t _a(n-1) pass between (seeing attached list 2) is thinking that the measured value of this standard substance is consistent with standard value, there is not the variation of conspicuousness in the characteristic value of this standard substance; Otherwise think that the variation of conspicuousness has occurred the characteristic value of this standard substance.

The study on the stability of fugutoxin the results are shown in Table 2.

Table 2 fugutoxin study on the stability result (μ g/kg) (20 DEG C)

The fugutoxin positive quality control sample of development, through 6 months study on the stability, is analyzed through t method of inspection in the measurement result of different time, its thinking that the measured value of this standard substance is consistent with standard value, there is not the variation of conspicuousness in the characteristic value of this standard substance.Study on the stability result shows that this positive quality control sample (4 DEG C) in 6 months has good stability.

5. definite value and uncertainty evaluation

Definite value

In JJG1006-1994, specify:

" homogeneity is qualified, and the satisfactory standard substance of stability test can carry out definite value.”

" select one of following manner to standard substance definite value: by the absolute or authoritative measuring method definite value of pin-point accuracy, by the reliable method definite value of the known accuracy of two or more principles, multiple laboratory cooperation definite values.”

" statistical treatment of value data:

When by " by the absolute or authoritative measuring method definite value of pin-point accuracy " mode definite value, measurement data can be by following routine processes.

To each operator's one group of independent measurement result, the generation of dubious value is described technically and gives after deletion, available Grubbs (Grubbs) method or Rod Dixon (Dixon) method are statistically rejected dubious value again.When numeric ratio disperses or when dubious value is many, conscientiously check measurement method, measuring condition and operating process.List each operator's measurement result: raw data, mean value, standard deviation, measurement number of times.

Grubbs method of inspection is applicable to check the consistance of many group measured value averages and rejects the average that peels off in many group measured values; Also can be used for checking one group of measured value consistance and reject the outlier in one group of measured value, method is as follows:

Have n group measured value, the average of every group of measured value is respectively wherein Largest Mean is designated as minimum mean is designated as

Calculate grand mean and standard deviation

$\stackrel{=}{x}=\frac{1}{n}\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}{\stackrel{=}{x}}_i,s_{\stackrel{\&OverBar;}{x}}=\sqrt{\frac{1}{n-1}\underset{i=1}{\overset{n}{\mathrm{\&Sigma;}}}{({\stackrel{\&OverBar;}{x}}_i-\stackrel{=}{x})}^2}$

Suspicious average is minimum value time, be calculated as follows statistic (T):

$T=\frac{\stackrel{=}{x}-{\stackrel{\&OverBar;}{x}}_{\min }}{s_{\stackrel{\&OverBar;}{x}}}$

Suspicious average is maximal value time, be calculated as follows statistic (T):

$T=\frac{{\stackrel{\&OverBar;}{x}}_{\max }-\stackrel{=}{x}}{s_{\stackrel{\&OverBar;}{x}}}$

According to measured value group number and given level of significance (α), check in critical value from Grubbs (Grubbs) inspection tables of critical values (seeing attached list 3);

If T≤T _0.05, suspicious average is normal mean value;

If T _0.05<T≤T _0.01, suspicious average is for departing from average;

If T>T _0.01, suspicious average is the average that peels off, and should reject, and rejects the one group of data that contains this average.

Average and the standard deviation of table 3 fugutoxin positive quality control sample determination value

Suspicious average is minimum value time, $T=\frac{\stackrel{=}{x}-{\stackrel{\&OverBar;}{x}}_{\min }}{s_{\stackrel{\&OverBar;}{x}}}=\frac{72.78-72.38}{0.18814}=2.073$

Suspicious average is maximal value time, $T=\frac{{\stackrel{\&OverBar;}{x}}_{\max }-\stackrel{=}{x}}{s_{\stackrel{\&OverBar;}{x}}}=\frac{73.12-72.78}{0.18814}=1.807$

T≤T _0.05(2.290), suspicious average is normal mean value.

The uncertainty evaluation of measurement result

According to ISO, EURACHEM directive/guide, evaluation of uncertainty in measurement and expression (national metering technology normalized), the uncertainty of the fugutoxin positive quality control sample to development is evaluated.

1. the uncertainty u that standard substance purity is brought ₁

Adopt liquid-phase chromatography method to measure the standard substance of development, obtain purity number percent with the peak area of target determinand divided by the total peak area of target determinand and impurity.

Table 4 fugutoxin concentration and purity

By convention, purity uncertainty is generally decided to be 0.3%, according to rectangular distribution, obtains divided by comprising the factor relative standard uncertainty that purity is introduced, and is designated as u ₁.

TTX： $u_1=\frac{0.30\%}{98.0\%\&times;\sqrt{3}}=0.18(0.17670\%)$

2. the uncertainty u that apparatus measures is brought ₂

It is definite that the repeatability of apparatus measures is repeatedly measured total relative standard deviation by instrument, and the uncertainty that apparatus measures is brought is designated as u ₂.

Table 5 apparatus measures relative standard deviation

The relative uncertainty u that 3 stability are brought ₃

Stability measurement result shows that value is stable in monitoring time, and the relative standard deviation of four each toxin determination values of time period is designated as u ₃.

Table 6 stability result relative standard deviation

4 uncertainty combinations

By each component of the uncertainty obtaining, by following formula, 4 components are synthetic, obtain fugutoxin standard substance combined standard uncertainty u _c.

$u_c=\sqrt{{u_1}^2+{u_2}^2+{u_3}^2}$

$u_c=\sqrt{{0.18\%}^2+{0.26\%}^2+{0.18\%}^2}=0.36\%$

Combined standard uncertainty is multiplied by and comprises the factor, obtains expanded uncertainty U, in table 7.

Table 7 Microcystin standard substance standard value and uncertainty

When degree of confidence is 99%, k generally gets 3

The above embodiment of the present invention is can not be used for limiting the present invention to explanation of the present invention, and any change in implication and the scope suitable with claims of the present invention, all should think to be included in the scope of claims.

Claims (8)

1. the preparation method of fugutoxin positive quality control sample, comprise positive is diluted and negative sample carried out to two kinds of standard substance interpolations and prepare scheme, it is characterized in that, comprise the following steps: scarlet fin dongfang globe fish to get homogeneous after musculature, measure the wherein content of fugutoxin, whether checking contains fugutoxin, if positive, the positive that contains toxin is diluted and mixed with negative sample and spreading agent, continue to measure the wherein content of toxin, reach after mark content requirement, by sample packing, under proper temperature, preserve, sample is carried out to homogeneity checking and stability analysis, if negative sample, adds fugutoxin standard solution in negative sample to, add spreading agent to mix, measure the wherein content of fugutoxin, reach after mark content requirement, by sample packing, under proper temperature, preserve, sample is carried out to homogeneity checking and stability analysis.

2. the preparation method of fugutoxin positive quality control sample according to claim 1, is characterized in that, specifically comprises the following steps:

(1) get scarlet fin dongfang globe fish and get musculature, homogenizer homogeneous, in homogenizing process, pressure is 25-35Mpa, homogenizing time adopts gradient Time Method, homogeneous three times altogether, each time is respectively 15-20 min, 10-15 min, 5-10 min;

(2) sample good homogeneous is carried out to toxin determination, adopt conventional method to measure, according to whether containing toxin, preparation method is divided into positive dilution and negative sample standard substance adds two kinds;

Positive dilution: the positive that contains toxin is mixed with negative sample and spreading agent dilution, spreading agent weight accounts for the 8-12% of positive and stealthy sample general assembly (TW), dilution mixes the stepwise dilution method that adopts, negative sample and spreading agent are divided into 3-5 part, after adding a negative sample and spreading agent, once, each homogenizing time is 3-5min to homogeneous;

Negative sample standard substance adds: fugutoxin standard solution is added to after measured not containing in the scarlet fin dongfang globe fish musculature of fugutoxin, then average mark adds for 3-5 time spreading agent to mix, often add a spreading agent homogeneous once, each homogenizing time is 3-5min, add the total amount of spreading agent to account for the 8-12% of negative sample total amount

(3) content of toxins of sample in determination step (2);

(4) content of toxins reaches after Identification Demand, and by sample packing, proper temperature is preserved, and sample is carried out to homogeneity checking and stability analysis.

3. the preparation method of fugutoxin positive quality control sample according to claim 2, is characterized in that, in step (1), musculature before homogeneous, is heated to 40-50 DEG C by musculature in homogenizer, and when homogeneous, homogenizing temperature is controlled at 30-40 DEG C.

4. the preparation method of fugutoxin positive quality control sample according to claim 2, is characterized in that, described spreading agent is C ₁₈, the combination of one or more in graphitized carbon, white carbon, zeyssatite.

5. the preparation method of fugutoxin positive quality control sample according to claim 2, is characterized in that, content of toxins reaches after Identification Demand, by sample freeze drying or oven dry, does powdering or wet-milling shape or pulpous state.

6. the preparation method of fugutoxin positive quality control sample according to claim 2, is characterized in that: sample storage condition is that 4 DEG C, low temperature or ultralow temperature are preserved.

7. a fugutoxin positive quality control sample that utilizes the preparation method of the fugutoxin positive quality control sample described in claim 2 to obtain.

8. fugutoxin positive quality control sample claimed in claim 7 is applied as detecting quality control in fugutoxin testing process.