CN103983495B - A kind of tetrodotoxin positive quality control sample and its preparation method and application - Google Patents

Abstract

The invention discloses the preparation method of a kind of tetrodotoxin positive quality control sample, step for taking homogenizing after muscular tissue by scarlet fin east, measure the content of wherein tetrodotoxin, verify whether containing tetrodotoxin, if positive, positive negative sample containing toxin and dispersant are diluted mixing, continue to measure the content of wherein toxin, after reaching requirement, by sample subpackage, preserve under proper temperature, sample is carried out uniformity and stability analysis；If negative sample, tetrodotoxin standard solution is added in negative sample, add dispersant mixing, measure the content of wherein tetrodotoxin, after reaching requirement, by sample subpackage, preserve under proper temperature, sample is carried out uniformity and stability analysis.Quality-control sample of the present invention preparation is simple, has enough stability and uniformity；Holding time is long, can be used for Good Laboratory control.

Description

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

Technical field

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

Background technology

East (including red fin east (Fugu rubripes) and Puffer fish (Fugu obscurus) etc.) is The most extensive artificial cultivation class fingerling, albumen and lipid content in the flesh of fish enrich, and are U.S. of preciousness after detoxification process Taste delicacies.In order to monitor Various Seasonal river fish toxicity, measure TTX content in river fish tissue quickly and accurately, distinguish poisonous River and nontoxic river, understand Difference In Toxicity and the situation of change of common river fish in real time, and the most necessary foundation is a kind of the most just Prompt, highly sensitive, workable TTX detection method, and prepare the tetrodotoxin positive quality control sample of practicality, can be at TTX Detect with actual sample during detection, to weigh the reliability of tetrodotoxin testing result in river fish substrate, to carrying simultaneously The edible safety of high river fish is significant to open domestic and international river fish market；Also for food safety, environment prison The measurement of the aspects such as survey, sanitary inspection quarantine and research work provide technical support and magnitude tracing guarantee.

River fish meat delicious, nutritious, eat raw, cook, process all preferably, but due to the tetrodotoxin containing severe toxicity (Tetrodotoxin, TTX), can not become the food on popular dining table so far.River poison, i.e. contain in the body of river is a kind of acute Poison nerve toxin, is also present in newt, speckle foot toad and gastropod body, is the most malicious non-of discovery in current nature One of protein substance, its toxicity is equivalent to 1250 times of poisonous drugs potassium cyanide, only needs 0.48mg can cause people desperately, with stone room Clam toxin, liguatoxin are collectively referred to as the big public hazards toxin in ocean three.Along with the development of fishery, river fish poisoning in recent years is repeatly Appear in the newspapers end.The poisoning of river fish and gastropod is eaten in Japan, China's Mainland, Taiwan Province and some countries in Southeast Asia Happen occasionally, especially eat the poisonings such as small-sized gastropod basket whelk in China and have been reported that more, and cause many people dead Die and become serious public health problem (P.A.Hwang et al, 2007).EC regulations " Regulation853/2004/ EC " specify that poisonous river fish and similar kind are forbidden in EU market sale with " Regulation 854/2004/EC ".

Positive quality control sample is that sample a kind of and to be measured is same or analogous, but confirms with traceability and have regulation to survey The material of amount uncertainty standard value, has the characteristic of material measure in terms of storage value and transmission value.Thus positive matter Control sample is for ensureing reliability and the effectiveness of testing result, it is ensured that examination criteria is implemented effectively at laboratory monitoring and had Important function.

Have tetrodotoxin standard substance, such as the tetrodotoxin standard sample of National Bureau of Oceanography the 3rd Research Institute at present Product (the actually purification standard substance without river fish periplast), but the extraction standard sample of tetrodotoxin there is not yet research preparation Report.

Owing to the character of tetrodotoxin is slightly soluble in water (toxin being contained in internal organs can be dissolved in water), ethanol, concentrated acid, it is soluble in Diluted acid, insoluble in any neutral organic solvent, including ether, methanol, ethanol, acetone, N, N '-dimethyl Methanamide, diformazan are sub- Sulfone, benzene, chloroform and acetonitrile etc..This physicochemical characteristics being not readily dissolved in any neutral organic solvent, and river fish The existence of the matrix effect that tissue is complicated so that the quantitative determination of tetrodotoxin is always food inspection, ecology and medical science One challenge of research field.For ensureing reliability and effectiveness, the most urgently matrix TTX positive quality control sample of testing result Development.

Summary of the invention

The technical problem to be solved is: provide a kind of matrix TTX positive quality control sample, described quality-control sample There is enough stability and uniformity.

For solving above-mentioned technical problem, the technical solution used in the present invention is:

The present invention provides the preparation method of a kind of tetrodotoxin positive quality control sample, including positive dilution and negative sample Product standard substance adds two schemes, comprises the following steps: to take scarlet fin east homogenizing after muscular tissue, measures wherein river The content of toxin, verifies whether containing tetrodotoxin, if positive, by the positive negative sample containing toxin With dispersant dilution mixing, continue to measure the content of wherein toxin, after reaching to identify content requirement, by sample subpackage, suitably temperature Degree is lower to be preserved, and sample is carried out uniformity and stability analysis；If negative sample, tetrodotoxin standard solution is added In negative sample, add dispersant mixing, measure the content of wherein tetrodotoxin, after reaching requirement, by sample subpackage, suitably At a temperature of preserve, sample is carried out uniformity and stability analysis.

The preparation method of tetrodotoxin positive quality control sample described above, specifically includes the following step:

(1) taking scarlet fin east and take muscular tissue, homogenizer homogenizing, in homogenizing process, pressure is 25-35Mpa, homogenizing Time uses gradient timetable method, altogether homogenizing three times, and each time is respectively 15-20min, 10-15min, 5-10min；

(2) sample good for homogenizing is carried out toxin determination, use conventional method to measure, according to whether containing toxin, Preparation method is divided into positive dilution and negative sample standard to add two kinds；

1. positive dilution: the positive negative sample containing toxin and dispersant are diluted mixing, dispersant Weight accounts for positive and the 8-12% of negative sample gross weight, and dilution mixing uses stepwise dilution method, by negative sample and point Powder is divided into 3-5 part, and after adding a negative sample and dispersant, homogenizing is once, and each homogenizing time is 3-5min；

2. negative sample standard is added: tetrodotoxin standard solution adds to the scarlet fin after measured without tetrodotoxin In the muscular tissue of east, then average mark adds dispersant mixing for 3-5 time, often adds a dispersant homogenizing once, the most all The matter time is 3-5min, and the total amount adding dispersant accounts for the 8-12% of negative sample total amount.

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

(4), after content of toxins reaches to identify content requirement, by sample subpackage, proper temperature preserves, carries out sample uniformly Property checking and stability analysis.

Preferably, in step (1), muscular tissue before homogenizing, is heated to 40-50 DEG C in homogenizer by muscular tissue, During homogenizing, homogenizing temperature controls at 30-40 DEG C.Muscular tissue keeps one in heating and homogenizing process before homogenizing in homogenizer Fixed temperature, can be preferably by toxin dissolution.

Described dispersant is C₁₈, a kind of or combination of two kinds in graphitized carbon, white carbon, kieselguhr.Add dispersant Sample substrate viscosity can be advantageously reduced, it is simple to TTX toxin is dispersed in substrate, make sample stable；Also with sample detection During pretreatment process keep consistent, there is during sample determination adsorbing contaminant, purify the feature of extracting solution.(effect Be all SP2 hydridization between the carbon atom on principle GCB surface, have single electron to and active ion species, and have hexagonal microcosmic knot Structure so that it has strong adsorption for planar molecule or the molecule containing plane aromatic rings, can remove and have altogether The impurity such as the phenylalanine of yoke structure, tyrosine, tryptophan and histidine；The aggregate performance of GCB surface is hydrophobicity, adsorbable non- Polarity and low pole compound；There are some polar sites and enable it to as ion-exchanger or absorption polar in its subsurface Compound, under this extraction conditions tested, adsorbable river fish flavor components glutamic acid, arginine, aspartic acid etc., show as The absorption property of wide spectrum；C₁₈Also it is a kind of nonpolar broad spectrum activity cleanser, it is possible to active adsorption extracts non polar impurities；And diatom Soil is then had sieving actoion, in-depth filtration and is reached by molecular separating force (Van der Waals force), Zeta potential and ion exchange The effect of adsorbing contaminant.)

Preferably, after step (4) content of toxins reaches to identify content requirement, can continue sample lyophilization or drying, Do 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 be respectively equivalent to 1g sample, 2g sample, 3g sample, 4g sample, 5g sample etc., in order to consistent with practical operation sample weighting amount.

Preferably, sample storage condition is 4 DEG C, low temperature or Excised Embryos.

The present invention also provides for a kind of preparation method utilizing described tetrodotoxin positive quality control sample and obtains tetrodotoxin Positive quality control sample.

The natural origin of quality-control sample mesostroma tetrodotoxin (TTX) of the present invention includes the toxic river that nature generates The living organism such as fish or basket whelk, but it is several to be not limited to this.

In described quality-control sample tetrodotoxin (TTX) source for tetrodotoxin (TTX) standard substance (include natural purification and Chemosynthesis).

The invention has the beneficial effects as follows:

Quality-control sample of the present invention preparation is simple, has enough stability and uniformity；Holding time is long, available In Good Laboratory control.The dispersant added have dispersion, stable, contribute to analyzing pretreatment process in detection program The functions such as extraction and cleaning.

Detailed description of the invention

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

(bare substrate standard addition method, formula is C with formulation Example 1 in preparation₁₈With graphitized carbon powder, this example adds Add agent C₁₈It is 1:1 with graphitized carbon ratio)

1) sampling Preliminary detection TTX contain the scarlet fin east measuring artificial cultivation, cut off back epidermis and take muscle portion Divide and avoid getting blood and internal organs, obtaining 400g muscular tissue, surveying with analyzing grinder (IKA A11 B25) homogenizing post analysis Fixed, result is not contain tetrodotoxin in this sample；2) mark-on mixing adds the standard solution containing 20 μ g TTX, mixing 15min；3) add dispersant and stabilizer average mark adds dispersant (C 3-5 time₁₈20g each with graphitized carbon powder) mixing, often Adding a dispersant homogenizing once, each homogenizing time is 3-5min, and the total amount adding dispersant accounts for negative sample total amount 10%, fully measure TTX content by HPLC-MS/MS method after mixing, measurement result is in tolerance interval, then mensuration is carried out Homogeneity；4) subpackage, frozen, stability inspection accurately weigh 1.10g sample and (are equivalent in 50ml polypropylene centrifuge tube 1.00g river fish meat sample), seal ,-20 degrees Celsius of preservations, with 1 month, 2 months, half a year, within 1 year, carry out for interval Stability is examined or check.

(bare substrate standard addition method, formula is C with formulation Example 2 in preparation₁₈With graphitized carbon powder, this example adds Add agent C₁₈It is 2:1 with graphitized carbon ratio)

1) sampling Preliminary detection TTX contain the scarlet fin east measuring artificial cultivation, cut off back epidermis and take muscle portion Divide and avoid getting blood and internal organs, obtaining 400g muscular tissue, surveying with analyzing grinder (IKA A11 B25) homogenizing post analysis Fixed, result is not contain tetrodotoxin in this sample；2) mark-on mixing adds the standard solution containing 20 μ g TTX, mixing 15min；3) add dispersant and stabilizer average mark adds dispersant (C 3-5 time₁₈With graphitized carbon powder respectively 26.7g and 13.3g) mixing, often adds a dispersant homogenizing once, and each homogenizing time is 3-5min, and the total amount adding dispersant accounts for feminine gender The 10% of sample total amount, fully measures TTX content by HPLC-MS/MS method after mixing, measurement result in tolerance interval, Measure again and carry out Homogeneity；4) subpackage, frozen, stability inspection accurately weigh 1.10g sample in 50ml polypropylene centrifuge Pipe (is equivalent to 1.00g river fish meat sample), seals, in-20 degrees Celsius of preservations, with 1 month, 2 months, half a year, one Year carries out stability examination for interval.

(formula is single addition of C to formulation Example 3₁₈Powder)

1) sampling Preliminary detection TTX contain the scarlet fin east measuring artificial cultivation, cut off back epidermis and take muscle portion Divide and avoid getting blood and internal organs, obtaining 400g muscular tissue, surveying with analyzing grinder (IKA A11 B25) homogenizing post analysis Fixed, result is not contain tetrodotoxin in this sample；2) mark-on mixing adds the standard solution containing 20 μ g TTX, mixing 15min；3) add dispersant and stabilizer average mark adds dispersant (C 3-5 time₁₈40g) mixing, often adds a dispersant equal Once, each homogenizing time is 3-5min to matter, and the total amount adding dispersant accounts for the 10% of negative sample total amount, fully uses after mixing HPLC-MS/MS method measures TTX content, and measurement result is in tolerance interval, then mensuration carries out Homogeneity；4) subpackage, Frozen, stability inspection accurately weighs 1.10g sample and (is equivalent to 1.00g river fish meat sample in 50ml polypropylene centrifuge tube Product), seal ,-20 degrees Celsius of preservations, with 1 month, 2 months, half a year, within 1 year, carry out stability examination for interval.

Formulation Example 4

Additive formulations is C₁₈Powder, additive formulations is C₁₈, graphitized carbon and kieselguhr ratio be 2:2；1, but not office It is limited to this.

Use embodiment 1

Sample pretreatment

1) river fish freezing of living is put to death, peeling, takes its muscle parts and goes muscle fully to pulverize through homogenizing sampling machine.

2) weigh sample (1.00 ± 0.01) g in 50mL polypropylene centrifuge tube, accurately add 1% acetic acid methanol (1:99, V/v) solution 5mL, homogenizing 30s, be centrifuged in 50 DEG C of supersound extraction 25min.8000r/min*5min, and transfer supernatant is clean to another In clean centrifuge tube；Residue 4mL 1% acetic acid methanol solution repeats to extract once, merges supernatant and in 4 DEG C of 12000r/min Centrifugal 5min, gained supernatant is to be clean；

3) take 1.0mL supernatant in 2mL EP (Eppendorf) pipe, add 100mg ketjenblack EC (GCB) and 100mg C₁₈Violent vortex mixed 30s of powder, 10000r/min is centrifuged 5min, and the supernatant is through 0.22 μm politef-Buddhist nun Dragon composite membrane is collected in plastics sample introduction bottle HPLC-MS/MS after filtering and measures.

Positive quality control sample mensuration takes the tetrodotoxin positive quality control sample of 50mL polypropylene centrifuge tube packaging and (is equivalent to river Flesh of fish substrate (1.00 ± 0.01) g), accurately add 1% acetic acid methanol (1:99, v/v) solution 5mL, homogenizing 30s, in 50 DEG C Supersound extraction 25min.8000r/min*5min is centrifuged, in transfer supernatant to another clean centrifuge tube；Residue 4mL 1% second Acid methanol solution repeats to extract once, merges supernatant and is centrifuged 5min in 4 DEG C of 12000r/min, and gained supernatant is to be clean； Remaining processes ibid.

Uniformity and stability test

Uniformity is investigated

By rule and statistical method (the GB/T 15000.3-of standard sample work directive/guide (3) standard sample definite value 2008/ISO Guide 35:2006) 5.8 carry out uniformity research, by 2 × N of total sample unit number N^1/3Randomly drawing sample.

By " mensuration of tetrodotoxin in the fresh river fish of GB/T5009.206-2007 " and " high performance liquid chromatography/tandem mass spectrum Method measures TTX in the tissue of river " detection, each sample retest 2 times, use method of analysis of variance (F-inspection) sample survey Uniformity between Ping.Sample homogeneity assay is shown in Table 1.

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

According to the sample homogeneity method of inspection, once calculate

M=13 N=26

Overall average

Sum of squares between groups

Quadratic sum in group

$Q_2=\underset{i=1}{\overset{m}{\Σ}}\underset{j=1}{\overset{n}{\Σ}}{(x_{ij}-\stackrel{\‾}{x_i})}^2=0.79250$

v₁=m-1=12

v₂=N-m=13

The amount of taking statistics F

$F=\frac{\frac{Q_1}{v_1}}{\frac{Q_2}{v_2}}=\frac{0.07103}{0.06096}=1.17$

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 variable and intra-class variance, the two ratio is less than the marginal value of statistical test, therefore tetrodotoxin is positive In property quality-control sample, the content distribution of TTX is uniform.

Study on the stability

JJG 1006-1994 specifies:

" standard substance under the conditions of the storage of regulation or using, should carry out the stability of characteristic magnitude undetermined termly and examine Test.”

" time interval of stability test can be by the closeest rear principle arrangement dredged.When should have multiple within the valid period Between interval Monitoring Data.”

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

" investigate stability specimen in use to randomly draw from the sample being distributed into minimum package unit, the sample of extraction Number has enough representativenesses for Bulk Samples.”

The stability of tetrodotoxin is monitored, in 6 months, respectively to the Stability Determination of tetrodotoxin 4 Secondary, take 3 tetrodotoxin samples the most at random, application t method of inspection carries out estimation of stability.

T method of inspection: when the standard value of standard substance characteristic magnitude is known, can use the steady of t method of inspection evaluation criterion material Qualitative, by formulae express be

$|{\stackrel{\‾}{X}}_i-\stackrel{\‾}{X}|\≤t_a(n-1)\·\frac{s}{\sqrt{n}}$

In formula,Average value measured for arbitrary standard material stability monitoring；For standard substance characteristic magnitude Standard value；t_a(n-1) it is that t checks marginal value；S is the standard deviation of measuring method；N is pendulous frequency.

IfMarginal value t is checked with t_a(n-1) (see attached list 2) between relation beThink The measured value of this standard substance is consistent with standard value, and the characteristic magnitude of this standard substance does not occur the change of significance；Otherwise Think that the characteristic magnitude of this standard substance there occurs the change of significance.

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

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

The tetrodotoxin positive quality control sample developed is examined through t through 6 months study on the stability, the measurement result at different time Test method analysis, itsThink that the measured value of this standard substance is consistent with standard value, the spy of this standard substance There is not the change of significance in property value.Study on the stability result shows this positive quality control sample (4 DEG C) stability in 6 months Well.

5. definite value and uncertainty evaluation

Definite value

JJG 1006-1994 specifies:

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

" select a pair standard substance definite value of following manner: by the absolute of high accuracy or authoritative measuring method definite value, use The reliable method definite value of the known accuracy of two or more principles, multiple laboratory cooperation definite values.”

" statistical disposition of value data "

When by " by the absolute of high accuracy or authoritative measuring method definite value " mode definite value, measurement data can be by following journey Sequence processes.

One group of independence measurement result to each operator, after the generation of dubious value being described technically and giving deletion, can Dubious value is the most again rejected by Grubbs (Grubbs) method or Rod Dixon (Dixon) method.When numeric ratio is relatively decentralized or When dubious value is more, should conscientiously check measurement method, measuring condition and operating process.List each operator's measurement result: Initial data, meansigma methods, standard deviation, pendulous frequency.

Grubbs Law be applicable to the concordance checking many group measured value averages and reject many group measured values from Group's average；Can also be used for the outlier checked one group of measured value concordance and reject in one group of measured value, method is as follows:

Having n group measured value, the average often organizing measured value is respectivelyWherein maximum equal Value is designated asMinimum mean is designated as

Calculate grand meanAnd standard deviation

$\begin{array}{cc}\stackrel{\‾}{\stackrel{\‾}{x}}=\frac{1}{n}\underset{i=1}{\overset{n}{\Σ}}{\stackrel{\‾}{\stackrel{\‾}{x}}}_i& s_{\stackrel{\‾}{x}}=\sqrt{\frac{1}{n-1}\underset{i=1}{\overset{n}{\Σ}}{({\stackrel{\‾}{x}}_i-\stackrel{\‾}{\stackrel{\‾}{x}})}^2}\end{array}$

Suspicious average is minimaTime, it is calculated as follows statistic (T):

$T=\frac{\stackrel{\‾}{\stackrel{\‾}{x}}-{\stackrel{\‾}{x}}_{\min }}{s_{\stackrel{\‾}{x}}}$

Suspicious average is maximumTime, it is calculated as follows statistic (T):

$T=\frac{{\stackrel{\‾}{x}}_{\max }-\stackrel{\‾}{\stackrel{\‾}{x}}}{s_{\stackrel{\‾}{x}}}$

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

If T≤T_0.05, the most suspicious average is normal mean value；

If T_0.05<T≤T_0.01, the most suspicious average is deviation average；

If T > T_0.01, the most suspicious average is the average that peels off, and should reject, and i.e. rejects one group of data containing this average.

The average of table 3 tetrodotoxin positive quality control sample measurements and standard deviation

Suspicious average is minimaTime,

Suspicious average is maximumTime,

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 measurement technical specification), to development The uncertainty of tetrodotoxin positive quality control sample evaluated.

1. uncertainty u that standard substance purity is brought₁

Use liquid-phase chromatography method that the standard substance developed is measured, with the peak area of target determinand divided by target The total peak area of determinand and impurity obtains percent purity.

Table 4 tetrodotoxin concentration and purity

By convention, purity uncertainty is typically set to 0.3%, according to distributed rectangular, obtains purity divided by Coverage factor The relative standard uncertainty introduced, is designated as u₁。

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

2. uncertainty u that apparatus measures is brought₂

The repeatability of apparatus measures is determined by the relative standard deviation that instrument repetitive measurement is total, and it is the most true that apparatus measures is brought Degree of determining is designated as u₂。

Table 5 apparatus measures relative standard deviation

The relative uncertainty degree u that 3 stability are brought₃

Stability measurement result shows that value is stable within the monitoring time, the phase of four time period each toxin determination values Standard deviation is designated as u₃。

Table 6 stability result relative standard deviation

4 uncertainty combinations

The each component of uncertainty that will obtain, as the following formula by 4 components synthesis, obtains the synthesis of tetrodotoxin standard substance 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 Coverage factor, obtains expanded uncertainty U, is shown in Table 7.

Table 7 Microcystin standard substance standard value and uncertainty

When confidence level is 99%, k typically takes 3

The above embodiment of the present invention is the description of the invention and cannot be used for limiting the present invention, with the right of the present invention Any change in implication that claim is suitable and scope, is all considered as being included within the scope of the claims.

Claims (7)

1. the preparation method of tetrodotoxin positive quality control sample, including being diluted positive and marking negative sample Quasi-material adds, it is characterised in that comprises the following steps: to take scarlet fin east homogenizing after muscular tissue, measures wherein river The content of toxin, verifies whether containing tetrodotoxin, if positive, by the positive negative sample containing toxin It is diluted mixing with dispersant, continues to measure the content of wherein toxin, reach to identify after content requirement, by sample subpackage ,-20 Degree Celsius preserve, sample is carried out uniformity and stability analysis；If negative sample, tetrodotoxin standard solution is added It is added in negative sample, adds dispersant mixing, measure the content of wherein tetrodotoxin, after reaching to identify content requirement, by sample Product subpackage ,-20 degrees Celsius of preservations, sample is carried out uniformity and stability analysis.

The preparation method of tetrodotoxin positive quality control sample the most according to claim 1, it is characterised in that specifically include down Row step:

(1) taking scarlet fin east and take muscular tissue, homogenizer homogenizing, in homogenizing process, pressure is 25-35Mpa, homogenizing time Using gradient timetable method, altogether homogenizing three times, each time is respectively 15-20 min, 10-15 min, 5-10 min；

(2) sample good for homogenizing is carried out toxin determination, use conventional method to measure, according to whether containing toxin, preparation Method is divided into positive dilution and negative sample standard substance to add two kinds；

Positive dilutes: the positive negative sample containing toxin and dispersant are diluted mixing, and dispersant weight accounts for Positive and the 8-12% of negative sample gross weight, dilution mixing uses stepwise dilution method, by average to negative sample and dispersant Being divided into 3-5 part, after adding a negative sample and dispersant, homogenizing is once, and each homogenizing time is 3-5min；

Negative sample standard substance adds: tetrodotoxin standard solution adds to the scarlet fin east after measured without tetrodotoxin In quadratus meat tissue, dispersant is divided into 3-5 part, adds portion every time, often add a dispersant homogenizing once, the most all The matter time is 3-5min, and the total amount adding dispersant accounts for the 8-12% of negative sample total amount,

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

(4) content of toxins reach mark require after, by sample subpackage ,-20 degrees Celsius of preservation, sample is carried out uniformity with Stability analysis.

The preparation method of tetrodotoxin positive quality control sample the most according to claim 2, it is characterised in that in step (1), Muscular tissue before homogenizing, is heated to 40-50 DEG C in homogenizer by muscular tissue, and during homogenizing, homogenizing temperature controls at 30-40 ℃。

The preparation method of tetrodotoxin positive quality control sample the most according to claim 2, it is characterised in that described dispersant For C₁₈, graphitized carbon, white carbon, the combination of one or more in kieselguhr.

The preparation method of tetrodotoxin positive quality control sample the most according to claim 2, it is characterised in that content of toxins reaches After requiring to mark, by sample lyophilization or drying, do powdering or wet-milling shape or pulpous state.

6. the tetrodotoxin that the preparation method of the tetrodotoxin positive quality control sample that a kind utilizes described in claim 2 obtains is positive Quality-control sample.

7. the conduct detection quality control during tetrodotoxin detects of the tetrodotoxin positive quality control sample described in claim 6 Application.