CN111122752A - Preparation method of tetrodotoxin component analysis standard substance - Google Patents
Preparation method of tetrodotoxin component analysis standard substance Download PDFInfo
- Publication number
- CN111122752A CN111122752A CN202010119461.6A CN202010119461A CN111122752A CN 111122752 A CN111122752 A CN 111122752A CN 202010119461 A CN202010119461 A CN 202010119461A CN 111122752 A CN111122752 A CN 111122752A
- Authority
- CN
- China
- Prior art keywords
- tetrodotoxin
- standard substance
- sample
- preparation
- analysis standard
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The invention discloses a preparation method of a tetrodotoxin component analysis standard substance, which takes puffer fish as a sample, and prepares candidate standard substances through the processes of cleaning, impurity removal, homogenization, quantitative mixing, freeze-drying, crushing, sieving, split charging, irradiation, preservation and the like. And carrying out minimum sampling quantity, uniformity and stability inspection on the packed standard substance to ensure that the packed standard substance meets the requirements of national standard substance characteristics. The preparation method can better retain the original components in the matrix, has better uniformity, stability and rehydration property and longer quality guarantee period, and fills the blank of the standard substance of the tetrodotoxin matrix at home and abroad; the matrix standard substance can effectively evaluate the detection capability and level of tetrodotoxin in relevant laboratories, can be used for quality control of qualitative and quantitative analysis of tetrodotoxin, ensures the accuracy of detection of tetrodotoxin in aquatic products, and protects the driving for food safety.
Description
Technical Field
The invention belongs to the technical field of metering, and particularly relates to a preparation method of a tetrodotoxin component analysis standard substance.
Background
Tetrodotoxin (TTX) is an alkaloid contained in puffer fish and other organisms, and has a molecular formula of C11H17O8N3The compound is an amino perhydro quinazoline type compound, is one of neurotoxins which are found to be the most toxic in nature, has the toxicity which is more than 1250 times higher than that of cyanide, and can cause death by 0.5 mg. For a long time, scholars at home and abroad have made a series of studies on the origin of tetrodotoxin. Currently, most expert scholars believe that the true source of TTX is not the animal itself, but rather it is aggregated through the food chain or produced by its commensal microorganisms. When the artificial puffer fish is artificially fed, the food chain of the puffer fish is changed by holding a water source and feeding the feed, so that the production of the tetrodotoxin can be effectively controlled, but the toxin content of the artificially cultured puffer fish also needs to be strictly monitored, and the eating safety is ensured.
With the deepening of toxicology, form and metabolic analysis of tetrodotoxin, tetrodotoxin in aquatic products is bound to be the key content of food safety detection attention, a detection technology matched with tetrodotoxin needs to be further matured and become a system, and the demand of matrix standard substances related to tetrodotoxin is more urgent. By now, none of the world's food matrix biotoxin standards include tetrodotoxin matrix standards, both in the literature and in international authoritative standards databases (e.g., international standards databases, COMAR, european union standards databases IRMM and ERM, National Institute of Standards and Technology (NIST), Canada National Research Council of Canada (NRCC), etc.).
Disclosure of Invention
One of the purposes of the invention is to provide a preparation method of a tetrodotoxin component analysis standard substance, which fills the blank of tetrodotoxin matrix standard substances at home and abroad, and the prepared standard substance has good uniformity and stability.
In order to achieve the purpose, the invention adopts the following specific technical scheme:
a preparation method of tetrodotoxin component analysis standard substance comprises the following steps:
s1, sample collection: collecting a positive sample of wild puffer fish as a matrix standard substance, and using cultured puffer fish as a negative sample;
s2 sample processing: respectively decomposing the positive sample and the negative sample into muscle and liver, weighing, shearing the tissue, and fully homogenizing;
s3. candidate preparation:
① determining tetrodotoxin contents in the positive and negative samples by liquid chromatography-tandem mass spectrometry, calculating the mass required by mixing the positive and negative samples according to the total mass and toxin content of the preset standard substances, and mixing the positive and negative samples according to the calculated value;
② stirring the mixed sample, and freeze drying;
③ pulverizing and sieving the dried block sample to obtain tetrodotoxin component analysis candidate;
s4, subpackaging and preserving the candidate: uniformly mixing the obtained candidates, randomly extracting at least 10 small samples, performing uniformity primary screening, and if the RSD of the content of the tetrodotoxin in the small samples is measured to be less than or equal to 5%, subpackaging the samples as tetrodotoxin component analysis standard substances, or continuing to prolong the uniform mixing time; the sample is subpackaged and sealed, sterilized by Co-60 gamma ray irradiation, and then frozen and stored at the temperature of-20 to-80 ℃.
S5, performance evaluation: the candidate prepared in step S4 was tested for minimum sample size, uniformity, and stability.
The preparation method of the tetrodotoxin component analysis standard substance selects wild puffer fish and cultured puffer fish as positive samples and negative samples respectively, selects livers and muscles of the puffer fish as matrixes, and obtains the standard substance with toxin content by mixing the positive samples and the negative samples, so that the standard substance matrix of the invention is completely from the puffer fish, has no any additive, avoids the interference of impurities during application, can better retain the original components in the matrix, improves the detection accuracy, evaluates the quality of the standard substance by carrying out minimum sampling quantity, uniformity and stability test on the prepared standard substance, and meets the requirements of primary standard substance technical specifications (JJJG 1006-94), and the preparation method of the invention is scientific and reliable.
Preferably, the minimum sampling amount and uniformity in step S5 are analyzed by F-test; stability includes short term stability at 25 ℃ and long term stability at 4 ℃.
Further, standard substance fixed value uncertainty was synthesized for the results of the tests for uniformity and stability, respectively.
Preferably, the specific procedures and process parameters of the freeze-drying in step S3 are as follows: the pre-freezing temperature is-25 to-30 ℃, and the pre-freezing time is 1 h; sublimation drying is set to be two stages, the sublimation temperature of the first stage is set to be-15 to-25 ℃, the sublimation time is 10 to 15 hours, the sublimation temperature of the second stage is set to be-4 to-8 ℃, and the sublimation time is 10 to 15 hours; the temperature of the desorption drying is set to be 10-20 ℃, and the desorption drying time is 5-10 h. The complete dehydration of the mixed sample is ensured through the processes of prefreezing, two-stage sublimation and resolution drying, the rapid deterioration in the later storage process is avoided, the stability is improved, and the shelf life is prolonged.
Further, before freeze-drying, the uniformly mixed tissue sample needs to be tiled, and the tiling thickness is 1-4 cm. After the mixed sample is tiled, pre-freezing-two-stage sublimation-analysis drying treatment is carried out, the tiling thickness is controlled, the internal dehydration difficulty of the sample is reduced, and the dehydration speed is effectively accelerated while the complete dehydration is ensured.
Preferably, in the step S3, the mixed sample is continuously stirred for at least 2 hours by using a rotary stirrer; and step S4, the candidate is mixed for at least 12h by using a three-dimensional mixer. The mixing time is strictly controlled, the good mixing effect is ensured, and the uniformity of the standard substance is improved.
Preferably, in the step S3, the mesh number is 75-100 meshes, so as to improve the homogenization degree of the sample and further improve the quality of the finished product.
Preferably, in the step S4, the sample is sealed, sterilized by Co-60 γ -ray irradiation, and then frozen for storage.
The second object of the present invention is to provide a tetrodotoxin composition analysis standard substance prepared by the above method.
The invention also aims to provide application of the tetrodotoxin component analysis standard substance, including verification of a tetrodotoxin detection method in aquatic products and assessment of detection capability and level of tetrodotoxin in laboratories.
The invention has the following beneficial effects:
the tetrodotoxin component analysis standard substance and the preparation method fill the blank of tetrodotoxin matrix standard substances at home and abroad, respond to metering development planning (2013-year 2020) issued by State hospitals, are simple and reliable in preparation method, and the obtained sample has better uniformity, stability and rehydration property and longer quality guarantee period. The standard substance can effectively evaluate the detection capability and level of tetrodotoxin in relevant laboratories, can be used for quality control of qualitative and quantitative analysis of tetrodotoxin, ensures the accuracy of detection of tetrodotoxin in aquatic products, and protects the driving for food safety.
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example 1
1 materials and methods
1.1 instruments, reagents
The instrument comprises the following steps: aquity H ultra high pressure liquid chromatograph, Vortex, Inc.; waters TQD triple quadrupole tandem mass spectrometer, Vorte, Inc., USA; Milli-Q ultra pure Instrument, Millipore, USA; vortex oscillators, Haedoff, Germany; high speed refrigerated centrifuge Sigma, Germany; 1ml pipette, German brand Brand
Reagent: spanish laboratory CIFGA s.a. pure standard substance of tetrodotoxin; immunoaffinity purification column Jiangsu American bioscience and technology limited; acetonitrile, methanol (both chromatographically pure) germany Merck; formic acid, acetic acid, ammonium formate (all chromatographically pure), 0.22 μm nylon needle filter membrane Shanghai Anpu experiment science and technology Co., Ltd; ultrapure water (prepared by Millipore water purifier); 50 mL centrifuge tubes, Corning, USA.
1.2 tetrodotoxin content determination method
According to the standard: GB 5009.206-2016 determination of tetrodotoxin in aquatic products by liquid chromatography-tandem mass spectrometry;
the principle of the method is as follows: extracting a sample by using 1% acetic acid-methanol solution, purifying by using an immunoaffinity column, determining by using a liquid chromatography-tandem mass spectrometry method, and adopting an external standard method;
a pretreatment process: accurately weighing 0.4g of sample (accurate to 0.001 g), adding 1.6 mL of water for redissolving, adding 20mL of 1% acetic acid-methanol solution, carrying out vortex oscillation for 15 min, centrifuging at 8000r/min for 5min, accurately transferring 1mL of supernatant, adding 10mL of LPBS solution for diluting, adjusting the pH value to be 7-8 by using sodium hydroxide solution (1 mol/L), and waiting for purification; discharging the PBS solution sealed in the immunoaffinity column at a natural flow rate, adding the solution to be purified, passing through the column at a flow rate of 1 drop/s, then rinsing with 10mL of water, draining, eluting with 4mL of acetic acid-methanol solution (2 + 98) and 1mL of water in sequence, passing the eluent through a 0.22 mu m organic phase microporous filter membrane, and then performing liquid chromatography-tandem mass spectrometry.
2 Standard substance sample preparation
According to the monitoring results of different types, different parts and different seasons of wild puffer fish in coastal areas of Zhoushan in China for years, the wild puffer fish in most types contain tetrodotoxin, the toxin content in ovary and liver parts is obviously higher than that in fish meat, and the content in spring seasons is higher than that in winter seasons. 500 kg of wild puffer fish was collected from fishermen in coastal areas of Zhoushan to prepare standard substances.
The specific process is as follows:
s1, sample collection: in spring seasons, wild puffer fish is collected as a positive sample of a matrix standard substance. Meanwhile, the cultivated puffer fish is purchased as a negative sample; after rapid freezing, the frozen product was sent to the laboratory.
S2 sample processing: thawing the samples respectively, cleaning with deionized water, sucking with filter paper, removing fish skin, viscera, fish bone, etc., decomposing out muscle and liver, weighing, cutting tissue, and homogenizing at high speed in a chopper for about 30 min until no obvious filamentous substance is present.
S3. candidate preparation:
① the method comprises determining tetrodotoxin content in positive and negative samples by liquid chromatography-tandem mass spectrometry, calculating the mass required by mixing the positive and negative samples according to the total mass and toxin content of preset standard substances, and mixing the positive and negative samples according to the calculated value, wherein the total mass of the standard substance is preset to 40kg and the content is 2500 mug/kg (wet weight), the TTX content of the positive sample is 11600 mug/kg in the preliminary test, the TTX content of the negative sample is 57 mug/kg., and the positive sample is about 8.47kg and the negative sample is about 31.53 kg.
② stirring the mixed sample with a rotary stirrer for more than 2 hr, and spreading the mixed tissue
The metal plate is cleaned, and the thickness of the metal plate is tiled by 2 cm; freeze-drying and dehydrating the sample: pre-freezing at-30 deg.C for 1h, and maintaining for 1 h; sublimation drying is preliminarily set to 2 stages, the sublimation temperature of the 1 st stage is set to-20 ℃, the sublimation time is 12h, the sublimation temperature of the 2 nd stage is set to-5 ℃, and the sublimation time is 12 h; setting the analysis drying temperature to 15 ℃, and analyzing and drying for 8 h;
③ pulverizing the dried block sample, and sieving with 80 mesh sieve to obtain tetrodotoxin component analysis candidate.
S4 preparation of standard substance: uniformly mixing the obtained candidate in a three-dimensional mixer for 12 hours again; randomly extracting 10 small samples from the uniformly mixed sample, and performing uniformity primary screening to obtain the tetrodotoxin contents (mu g/kg) of the 10 small samples of 10497.2, 10625.8, 10836.5, 10714.7, 11664.7, 11047.4, 11360.4, 11642.7, 11460.9 and 11487.7 respectively, wherein RSD is less than or equal to 5%; the sample is filled into brown polyethylene bottles by the mass of 5g per bottle, and is frozen and stored at the temperature of minus 20 ℃ after being sealed in a spiral way (in order to ensure the stability of the TTX standard substance in the globefish meat and prevent the deterioration of the TTX standard substance, all packages are sterilized by Co-60 gamma ray irradiation, and the irradiation dose is 5 KGy).
S5, performance evaluation: the standard substance prepared in step S4 is subjected to minimum sampling amount, uniformity and stability test,
the specific methods and results are as follows:
① minimum sample size
According to the specifications of the primary standard substance technical specification (JJG 1006-94), the general standard of the standard substance definite value and the requirements of the statistical principle (JJF 1343-. In the same bottle of sample, 0.2g, 0.4g and 0.8g were weighed simultaneously, and for the same sampling amount, 9 parts were taken for the parallelism detection. The test results were analyzed using one-way analysis of variance (F-test): calculating the value F, and searching a F distribution critical value Fa, wherein when the sample F is less than Fa, the set sampling amount has no significant influence on the detection result; when the sample F > Fa, the set sampling amount is shown to have a significant influence on the detection result, so that the minimum sampling amount is determined. The results of the minimum sample size test are shown in Table 1.
TABLE 1 measurement of tetrodotoxin content/(in. mu.g/kg) at different sampling volumes
As can be seen from the test results, the sampling amounts of 0.2g, 0.4g and 0.8g have no significant influence on the test results. However, the smaller the sample weight, the greater the effect on the uniformity. In consideration of the whole detection process, the minimum sampling quantity is set to be 0.4g, and subsequent uniformity and stability tests are carried out according to the weighing quantity of 0.4 g.
② uniformity test
According to the general criterion of the constant value of the standard substance and the specification of the statistical principle (JJF 1343-. The sample detection sequence is strictly detected according to a preset random sequence in a sample feeding mode, so that the time variation (drift in the measurement process) of a measurement system is prevented from interfering with uniformity evaluation. The test results were scored using one-way analysis of variance (F-test): and calculating the value F, and checking a F distribution critical value Fa, wherein when the sample F is less than Fa, the sample shows that the significant difference between the samples does not exist, and the sample is uniform. The results of the uniformity test are shown in Table 2.
TABLE 2 TTX homogeneity assay detection and statistics in puffer fish
③ stability test
Stability is one of the important parameters of a standard substance, which refers to the property of a standard substance to maintain a characteristic quantity within a specified range at specified time intervals and under ambient conditions. The stability of a standard substance is used to describe the change in the characteristic quantity of the standard substance over time. The longer the specified time interval, the better the stability of the standard substance. The short term stability under simulated transport conditions and the long term stability under storage conditions of the standard substances developed are examined in this patent.
a. Short term stability test
Short term stability testing takes the form of a simultaneous stability assessment to examine the stability of the sample at 25 ℃ over 14 days. According to the principle of first densification and then thinning, the samples placed in the environment temperature of 25 ℃ are transferred to the reference temperature condition (-20 ℃) for continuous storage after 0, 1, 3, 7 and 14 days respectively. Taking 3 unit samples at each time point, detecting all 15 unit samples at the same time, taking 1 subsample for each unit, detecting according to a '1.2 analysis method' and carrying out short-term stability detection. The results of the test are shown in Table 3.
TABLE 3 short term stability test analysis results (mug/kg) for standard substances
The results obtained were statistically analyzed by a straight line fitting method, and the results are shown in table 4. I b 1/S (b1) I < t (0.95, 13), which indicates that the developed standard substance does not change significantly during transportation in a short period. It is undesirable for the amount of standard substance to change during transport, so that the uncertainty it causes is negligible.
TABLE 4 statistical results of short term stability of standard substances (mug/kg)
b. Long term stability test
The long-term stability test adopts a synchronous stability evaluation mode to investigate the stability of the sample at 4 ℃ within 6 months. According to the principle of first dense and then sparse, the samples placed in the environment temperature of 4 ℃ are respectively transferred to the reference temperature condition (-20 ℃) for continuous storage after 0, 1, 3 and 6 months. Taking 4 unit samples at each time point, detecting all 16 unit samples at the same time, taking 1 subsample for each unit, detecting according to a 1.2 analysis method, and performing long-term stability detection. The results of the test are shown in Table 5.
TABLE 5 statistical results of short term stability of standard substances (mug/kg)
The results obtained were statistically analyzed by a straight line fitting method, and the results are shown in Table 6. I b 1/S (b1) | < t (0.95, 13), which indicates that the developed standard substance is stable under the condition of 4 ℃ and is protected from light for 6 months, and the relative uncertainty caused by the standard substance is 4.29%. TABLE 6 Long term stability results for standard substances (μ g/kg)
In summary, the invention relates to a preparation method of a standard substance for analyzing tetrodotoxin components in puffer fish tissues, which takes wild puffer fish, fish meat for raising the puffer fish and fish liver as raw materials, and prepares the uniform and stable standard substance by the processes of cleaning, peeling, homogenizing, freeze-drying, crushing, sieving, irradiating, preserving and the like.
The above-mentioned embodiments are illustrative of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications of the present invention using the design concept should be covered by the claims of the present invention.
Claims (10)
1. A preparation method of a tetrodotoxin component analysis standard substance is characterized by comprising the following steps: the method comprises the following steps:
s1, sample collection: collecting a positive sample of wild puffer fish as a matrix standard substance, and using cultured puffer fish as a negative sample;
s2 sample processing: respectively decomposing the positive sample and the negative sample into muscle and liver, weighing, shearing the tissue, and fully homogenizing;
s3. candidate preparation:
① determining tetrodotoxin contents in the positive and negative samples by liquid chromatography-tandem mass spectrometry, calculating the mass required by mixing the positive and negative samples according to the total mass and toxin content of the preset standard substances, and mixing the positive and negative samples according to the calculated value;
② stirring the mixed sample, and freeze drying;
③ pulverizing and sieving the dried block sample to obtain tetrodotoxin component analysis candidate;
s4 preparation of standard substance: uniformly mixing the obtained candidates, randomly extracting at least 10 small samples, performing uniformity primary screening, and if the RSD of the content of the tetrodotoxin in the small samples is measured to be less than or equal to 5%, subpackaging the samples as tetrodotoxin component analysis standard substances, or continuing to prolong the uniform mixing time; the sample is subpackaged and sealed, and is frozen and preserved at the temperature of between 20 ℃ below zero and 80 ℃ below zero,
s5, performance evaluation: the standard substance prepared in step S4 was subjected to minimum sampling amount, uniformity, and stability test.
2. The method for preparing tetrodotoxin composition analysis standard substance as set forth in claim 1, comprising the steps of: analyzing the minimum sampling amount and the uniformity by adopting an F test in the step S5; stability includes short term stability at 25 ℃ and long term stability at 4 ℃.
3. The method for preparing tetrodotoxin composition analysis standard substance as set forth in claim 2, wherein: and synthesizing the uncertainty of the fixed value of the standard substance according to the detection results of the uniformity and the stability.
4. The method for preparing tetrodotoxin composition analysis standard substance as set forth in claim 1, comprising the steps of: the specific procedures and process parameters of the freeze-drying in step S3 are as follows: the pre-freezing temperature is-25 to-30 ℃, and the pre-freezing time is 1 h; sublimation drying is set to be two stages, the sublimation temperature of the first stage is set to be-15 to-25 ℃, the sublimation time is 10 to 15 hours, the sublimation temperature of the second stage is set to be-4 to-8 ℃, and the sublimation time is 10 to 15 hours; the temperature of the desorption drying is set to be 10-20 ℃, and the desorption drying time is 5-10 h.
5. The method for preparing tetrodotoxin composition analysis standard substance as set forth in claim 4, wherein: before freeze drying, the uniformly mixed tissue sample needs to be tiled, and the tiling thickness is 1-4 cm.
6. The method for preparing tetrodotoxin composition analysis standard substance as set forth in claim 1, comprising the steps of: step S3, continuously stirring for at least 2h by adopting a rotary stirrer in the process of uniformly mixing the mixed sample; and step S4, the candidate is mixed for at least 12h by using a three-dimensional mixer.
7. The method for preparing tetrodotoxin composition analysis standard substance as set forth in claim 1, comprising the steps of: in step S3, the mesh number is 75-100 meshes.
8. The method for preparing tetrodotoxin composition analysis standard substance as set forth in claim 1, comprising the steps of: and in the step S4, the sample is subpackaged and sealed, sterilized by Co-60 gamma ray irradiation and then frozen for storage.
9. The tetrodotoxin analysis standard substance prepared by the preparation method according to any one of claims 1-8.
10. The use of the tetrodotoxin composition analysis standard substance as set forth in claim 9, including the verification of tetrodotoxin detection methods in aquatic products and the laboratory assessment of tetrodotoxin detection ability and level.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010119461.6A CN111122752B (en) | 2020-02-26 | 2020-02-26 | Preparation method of tetrodotoxin component analysis standard substance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010119461.6A CN111122752B (en) | 2020-02-26 | 2020-02-26 | Preparation method of tetrodotoxin component analysis standard substance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111122752A true CN111122752A (en) | 2020-05-08 |
| CN111122752B CN111122752B (en) | 2022-06-10 |
Family
ID=70493145
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010119461.6A Active CN111122752B (en) | 2020-02-26 | 2020-02-26 | Preparation method of tetrodotoxin component analysis standard substance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111122752B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113419015A (en) * | 2021-07-26 | 2021-09-21 | 国家食品安全风险评估中心 | Preparation method and application of natural tetrodotoxin standard sample based on puffer fish base material |
| CN117433865A (en) * | 2023-12-21 | 2024-01-23 | 云南省计量测试技术研究院 | Heavy metal-containing puer raw tea matrix standard substance and preparation method thereof |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5807865A (en) * | 1995-10-24 | 1998-09-15 | Merck Sharp & Dohme Limited | 3,3-disubstituted piperidine derivatives and their use as therapeutic agents |
| US20020099226A1 (en) * | 2000-11-22 | 2002-07-25 | Maoqing Zhou | Method of purifying tetrodotoxin |
| CN1568999A (en) * | 2003-07-14 | 2005-01-26 | 南宁枫叶药业有限公司 | Stable freeze dried formulation of spheroidine for medical use |
| CN101475575A (en) * | 2009-02-05 | 2009-07-08 | 王维国 | Extraction of tetraodontoxin, and preparation thereof used in drug dependence of dolantin |
| CN103983495A (en) * | 2014-06-10 | 2014-08-13 | 山东出入境检验检疫局检验检疫技术中心 | Tetrodotoxin positive quality control sample as well as preparation method and application thereof |
| US20160083730A1 (en) * | 2013-04-18 | 2016-03-24 | Bar-Ilan University | Systems comprising non-immunogenic and nuclease resistant nucleic acid origami devices for molecular computation |
| US20160290998A1 (en) * | 2006-06-12 | 2016-10-06 | Robert C. Leif | Reagent system and method for modifying the luminescence of lanthanide (iii) macrocyclic complexes |
| CN106596790A (en) * | 2016-12-28 | 2017-04-26 | 山东出入境检验检疫局检验检疫技术中心 | Method for rapidly detecting content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry |
| CN107641128A (en) * | 2017-10-09 | 2018-01-30 | 浙江省海洋水产研究所 | A kind of method of high efficiency extraction tetraodotoxin |
| CN107957473A (en) * | 2018-01-12 | 2018-04-24 | 杨洁 | A kind of quick determination method of tetraodotoxin |
| CN109142602A (en) * | 2018-07-27 | 2019-01-04 | 中国水产科学研究院南海水产研究所 | A kind of preparation method of tetraodotoxin standard biological sample |
-
2020
- 2020-02-26 CN CN202010119461.6A patent/CN111122752B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5807865A (en) * | 1995-10-24 | 1998-09-15 | Merck Sharp & Dohme Limited | 3,3-disubstituted piperidine derivatives and their use as therapeutic agents |
| US20020099226A1 (en) * | 2000-11-22 | 2002-07-25 | Maoqing Zhou | Method of purifying tetrodotoxin |
| CN1568999A (en) * | 2003-07-14 | 2005-01-26 | 南宁枫叶药业有限公司 | Stable freeze dried formulation of spheroidine for medical use |
| US20160290998A1 (en) * | 2006-06-12 | 2016-10-06 | Robert C. Leif | Reagent system and method for modifying the luminescence of lanthanide (iii) macrocyclic complexes |
| CN101475575A (en) * | 2009-02-05 | 2009-07-08 | 王维国 | Extraction of tetraodontoxin, and preparation thereof used in drug dependence of dolantin |
| US20160083730A1 (en) * | 2013-04-18 | 2016-03-24 | Bar-Ilan University | Systems comprising non-immunogenic and nuclease resistant nucleic acid origami devices for molecular computation |
| CN103983495A (en) * | 2014-06-10 | 2014-08-13 | 山东出入境检验检疫局检验检疫技术中心 | Tetrodotoxin positive quality control sample as well as preparation method and application thereof |
| CN106596790A (en) * | 2016-12-28 | 2017-04-26 | 山东出入境检验检疫局检验检疫技术中心 | Method for rapidly detecting content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry |
| CN107641128A (en) * | 2017-10-09 | 2018-01-30 | 浙江省海洋水产研究所 | A kind of method of high efficiency extraction tetraodotoxin |
| CN107957473A (en) * | 2018-01-12 | 2018-04-24 | 杨洁 | A kind of quick determination method of tetraodotoxin |
| CN109142602A (en) * | 2018-07-27 | 2019-01-04 | 中国水产科学研究院南海水产研究所 | A kind of preparation method of tetraodotoxin standard biological sample |
Non-Patent Citations (3)
| Title |
|---|
| PAULA ABAL等: "Acute Oral Toxicity of Tetrodotoxin in Mice: Determination of Lethal Dose 50 (LD50) and No Observed Adverse Effect Level (NOAEL)", 《TOXINS》 * |
| 方科益: "水产品中11种海洋生物毒素的高效液相色谱-四极杆/静电场轨道阱高分辨质谱检测方法研究", 《分析测试学报> * |
| 郑仁锦等: "超高效液相色谱-电喷雾串联质谱法测定河豚鱼干中的河豚毒素", 《预防医学论坛》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113419015A (en) * | 2021-07-26 | 2021-09-21 | 国家食品安全风险评估中心 | Preparation method and application of natural tetrodotoxin standard sample based on puffer fish base material |
| CN117433865A (en) * | 2023-12-21 | 2024-01-23 | 云南省计量测试技术研究院 | Heavy metal-containing puer raw tea matrix standard substance and preparation method thereof |
| CN117433865B (en) * | 2023-12-21 | 2024-04-12 | 云南省计量测试技术研究院 | Heavy metal-containing puer raw tea matrix standard substance and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111122752B (en) | 2022-06-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102288464B (en) | Preparation method for standard sample of chloramphenicol residual lyophiled powder in muscle of carp | |
| CN111122752B (en) | Preparation method of tetrodotoxin component analysis standard substance | |
| CN102079788B (en) | Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof | |
| CN102323122A (en) | Preparation method of sulfamethoxazole residue freeze-dried powder standard sample in carp muscle | |
| CN107607645B (en) | Method for simultaneously detecting residues of various veterinary drugs in fish meat | |
| CN102323123A (en) | Preparation method of ciprofloxacin and enrofloxacin residue freeze-dried powder standard sample in carp muscle | |
| CN106770802B (en) | Method and kit for simultaneously detecting multiple vitamins in dry blood filter paper sheet | |
| CN109883782A (en) | A kind of milk powder standard substance and preparation method thereof containing melamine | |
| CN111579320A (en) | Egg standard substance containing chloramphenicol, preparation method and application | |
| CN104360052B (en) | A kind of Clenbuterol detection Quality Control animals urine dry powder and preparation method and application | |
| Barker et al. | Effects of estrogens on gametogenesis and steroid levels in the ovaries and pyloric caeca of Sclerasterias mollis (Echinodermata: Asteroidea) | |
| CN107741502A (en) | Carbon, the preparation method of nitrogen standard of stable isotope sample in a kind of beef protein | |
| Spencer et al. | A rapid simplified bioassay for somatomedin | |
| CN101936986B (en) | Method for detecting diazepam and chemoluminescence immunoassay kit special for same | |
| CN115628958A (en) | Egg powder matrix standard sample containing rimantadine and preparation method thereof | |
| CN111983107A (en) | Method for evaluating in vivo bioavailability of perchlorate in food matrix | |
| CN112063683B (en) | Method and kit for detecting antibiotic residues in food and production and circulation processes thereof | |
| CN114878276A (en) | Preparation method and analysis method of OA and DTX1 standard sample of scallop and Prorocentrum limerianum mixed matrix | |
| CN114414321A (en) | Fipronil sulfone residue analysis standard substance candidate in egg powder and preparation method thereof | |
| CN112526024A (en) | Method for detecting sulfonamide antibiotics and acetylated metabolites thereof in aquatic products | |
| CN112051343A (en) | Method for determining antibiotic residues | |
| CN110208427A (en) | A kind of Spanish mackerel Freshness evaluation method based on biogenic amine | |
| CN112903874B (en) | Free and conjugated clenbuterol standard substance contained in pig urine and preparation method thereof | |
| CN112903873B (en) | Free-state and conjugated-state salbutamol standard substance contained in swine urine freeze-dried powder after animal metabolism and preparation method thereof | |
| CN112683613B (en) | Preparation method of ivermectin matrix standard substance in goat milk |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |