CN101813583A - Method for pre-treating animal sample for stable isotope detection - Google Patents
Method for pre-treating animal sample for stable isotope detection Download PDFInfo
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Abstract
The invention discloses a method for pre-treating an animal sample for stable isotope detection. The method comprises the following steps: putting the animal sample into a utensil without stable isotope interference and freeze-drying the animal sample, and then grinding the animal sample into powder by using the utensil without stable isotope interference so as to finish the pretreatment for detecting the stable isotope in the animal sample. According to the method, a ceramic mortar, scissors or a cutter without stable isotope interference is selected to cut and grind the animal sample, and the freeze-drying step is used to replace a hot drying step in the existing method to reduce the exchange between the stable isotope in the hot drying air and the same stable isotope in the sample. When the detection of the stable isotope is performed after the animal sample is pre-treated by the method, the stable isotope detection result is accurate, and multiple detection results have small coefficient of variation, so the method provided by the invention is a simple, accurate and effective pre-treatment method for the stable isotope detection of the animal sample and is favorable for improving efficiency and accuracy of the stable isotope detection in agricultural products.
Description
Technical field
The present invention relates to a kind of pre-treating method that is used for the animal sample of stable isotope detection.
Background technology
But the traceability of product is the prerequisite of implementation issue product recall, and therefore, but the traceability of agricultural product obtains manufacturing enterprise and consumer's concern.Along with new " agricultural product quality and safety method " issuing and implementation, China will progressively carry out agricultural product quality and safety and review system, and the agricultural product of selling on the market that do not meet the quality safety standard are traced to its source, and find out responsibility, handle in accordance with the law.But omnidistance record-keeping system is the two big bases of realizing the agricultural product traceability with the detection technique of tracing to the source.China's agricultural production mainly is the unit with the family, the small scale One's name is legion, and marketing of agricultural products is managed also based on the free market, and the intermediate link from the farmland to the dining table is many.Therefore, implement very difficulty of omnidistance record-keeping system, also unrealistic.The technology of tracing to the source of carrying out the product place of production or certain safe composition by detection seems particularly important.The effect of carrying out the traceability detection has following four aspects: 1, the authenticity of place of production product has been indicated in checking, forms the parallel techniques of checking each other with the bar code traceability system; 2, misidentify mark or fake products; 3, distinguish the 3rd state () product; 4, examination has special mark for example " organic ", " green ", " ecology " product.
The detection technique of tracing to the source is exactly to review kind, feeding regime and the geographic origin of product and the process that product is experienced by some physics, chemistry, biological method in processing and storage.Be characterized in not knowing to obtain the information of each link under the prerequisite of background.At present, the topmost detection technique of tracing to the source in stable isotope carbon, nitrogen, hydrogen, the oxygen analysis.Different stable isotope relative abundances reflects the different characteristic of agricultural product, for example, stablizing hydrogen and oxygen isotope abundance has reflected the material evaporation, has concentrated and geological information such as precipitation, stable carbon isotope has reflected the photosynthesis of plants classification, and the stable nitrogen isotopes abundance has disclosed plant growth apart from the position of the tropic etc.A lot of research report shows, by the analysis of stable isotope, information such as the place of production that can agricultural product, feed, formation, carry out product trace to the source or authenticity is differentiated.China has formulated national standards such as " assay method of c4 plant sugar-stable carbon isotope rule of three in the honey " in 2002, the management of for China honey being mingled on analytical technology provides technique guarantee.But the pre-treatment program of these detection methods is long, and routine analyzer needs about 12 hours; And, when altogether the shearing, drying, homogenate etc. of plant sample are handled for solid, there is the pollution of ironware, cause problems such as testing result distortion.
Summary of the invention
The purpose of this invention is to provide a kind of pre-treating method that is used for the animal sample of stable isotope detection.
The pre-treating method that is used for the animal sample of stable isotope detection provided by the invention comprises the steps:
Animal sample is contained in the utensil that no stable isotope disturbs after the freeze drying, and the utensil grind into powder that disturbs with no stable isotope is finished the described pre-treating method that is used for the animal sample of stable isotope detection again.
In the said method, the utensil of utensil that described no stable isotope disturbs for constituting by stupalith, the described utensil that is made of stupalith is mortar, cuts or cutter, the utensil of selecting for use stupalith to constitute, can avoid the interference that isotope causes testing result in the utensil (as ironware) in the stable isotope detection, the various utensils that are made of stupalith all are applicable to this method.In the described freeze drying step, temperature is-25 ℃ to-65 ℃, preferred-40 ℃, the time with the sample bone dry till; The order number of described powder was a 20-40 purpose aperture sieve, preferred mistake 40 purpose aperture sieve.In this method, described stable isotope is carbon stable isotope and nitrogen stable isotope.In addition, for the bigger animal sample of volume, can be earlier with the described utensil that constitutes by stupalith (as cutting or cutter) to as described in animal sample cut into slices and shred, carry out follow-up freezing and dried again.Various freeze drying plants commonly used such as freeze drying equipment all are applicable to this method as shown in Figure 2.
Pre-treatment when this method is particularly useful for stable isotope in animal muscle sample, animal's liver sample or the animal body fluid sample and detects.When described animal sample is the animal body fluid sample, after the step of the described utensil grind into powder that disturbs with no stable isotope, also carry out following processing: described powder is dipped in carries out degreasing in the ethylene dichloride, filter drying.In the described defatting step, the time is 1-2 hour, and preferred 1 hour, temperature was 55-65 ℃, preferred 60 ℃, remove by filter impurity, get filtrate, in the described drying steps, temperature is for being higher than 50 ℃, preferred 40 ℃, the time with the sample bone dry till.
The present invention selects ceramic mortar that no stable isotope disturbs for use, cuts or cutter, animal sample is cut and grinds, and with the heated drying step in the existing method of freeze drying replacement, with the isotopic exchange of same stable in stable isotope and the sample in the minimizing heated drying air.When utilizing this method that animal sample is carried out the isotopic detection of the laggard line stabilization of pre-treatment, the stable isotope testing result is accurate, the repeated detection coefficient of variation as a result is little, thereby, method provided by the invention is the pre-treating method of a kind of simple animal sample stable isotope accurately and effectively when detecting, and helps to improve stable isotope detects in the agricultural product efficient and accuracy.
Description of drawings
Fig. 1 for sample cutting in the embodiment of the invention or when grinding used ceramic mortar, pottery cut and Stupalox, be followed successively by ceramic mortar from left to right, pottery is cut and Stupalox.
Fig. 2 is used freeze drying equipment in the pre-treating method of the present invention.
Embodiment
The present invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
The pre-treatment of embodiment 1, solid animal tissue sample
1) will provide by European Union-food research project of tracing to the source, contract number is after two parts of reference sample beef (being respectively reference sample 1 and reference sample 2) of 006942 thaw slightly, at least the described reference sample 1 of 50g and reference sample 2 are cut into the thin slice that thickness is 2mm with each with Stupalox, be contained in and be placed in the low temperature refrigerator in the pottery, take out after freezing 60 minutes in-80 ± 5 ℃, dry down with freeze drier shown in Figure 2 again at-40 ℃, make described reference sample 1 and reference sample 2 bone dries (as sample bone dry not, touch the pottery that holds sample with hand, sensation is than all the other article colds on every side).After drying is finished, described reference sample 1 and the ceramic scissors of reference sample 2 usefulness are shredded, grind with ceramic mortar again, make powder cross 40 order aperture sieve, the pre-treatment operation the when stable isotope of finishing described reference sample 1 and reference sample 2 detects.
Will with last identical beef sample reference sample 1 and reference sample 2, (this method is: beef sample is rubbed through meat grinder, at 70 ℃ of freeze-day with constant temperature 48h, pulverize with comminutor to use bibliographical information respectively.) method and the pre-treating method that provided of existing national standard (GB/T18932.1-2002) carry out pre-treatment, each sample determination 10 times, each 2 parallel samples.The carbon isotope of described reference sample 1 and reference sample 2 beef and nitrogen isotope are measured assignment jointly by 6 isotope analysis laboratories of European Union.
With the beef sample that three kinds of different pre-treating methods of above-mentioned usefulness dispose, measure the wherein relative abundance of carbon, nitrogen stable isotope according to following method.
1) mensuration program:
Get the 1mg sample, the tinfoil paper cup is wrapped the back and is delivered in the elemental analyser (Flash EA1112 type) by automatic sampler, and carbon in this sample and nitrogen element are converted into pure CO
2Gas and N
2Behind the gas, be the dilution instrument of ConfloIII type through model again, enter DELTAPlus Thermo Finnigan mass spectrometer at last and detect.
Concrete running parameter is as follows:
Elemental analyser: the oxidation furnace temperature is 900 ℃, 650 ℃ of reduction furnace temperature, and injector helium purge flow is 300ml/min, oxygen flow is 240ml/min, reference gas flow 200ml/min.
ConfloIII type dilution instrument: helium pressure is 0.6bar, CO
2Reference gas pressure is bar, N
2Reference gas pressure is 1.0bar.
DELTAPlus Thermo Finnigan mass spectrometer: the analytical column temperature is 40 ℃; Demarcate CO with USGS24 (δ 13CPDB=-16.00 ‰)
2Steel cylinder is demarcated N with IAEAN1 (δ 15Nair=0.4 ‰)
2Steel cylinder.With the steel cylinder gas of demarcating as standard.
2) result calculates:
Generally acknowledge in the world and use relative quantity to represent isotopic enrichment degree, computing formula is:
δ ‰=(R sample/R standard-1) * 1000
Wherein, stable carbon, nitrogen isotope ratio are used δ respectively
13C ‰ and δ
15N ‰ expression, R is the abundance ratio of heavy isotope and light isotope in the sample, promptly
13C/
12C and
15N/
14N.International standard substance is the plan belemnite (PDB) of Cretaceous System during South Carolina, United States PeeDee builds to carbon isotope, is atmospheric nitrogen to nitrogen isotope.
3) measurement result:
According to step 2) computing formula, by computer mass spectrometric spectrogram result is calculated according to blas, the measurement result of gained carbon isotope and nitrogen isotope respectively as shown in Table 1 and Table 2.
The result that table 1, carbon isotope are measured
α
*Expression conspicuousness detection level, different lowercases represents to have significant difference, identical lowercase is represented there was no significant difference; α represents utmost point conspicuousness detection level, and different capitalizations represents to have utmost point significant difference, and identical capitalization is represented there was no significant difference.
The result that table 2, nitrogen isotope are measured
α
*Expression conspicuousness detection level, different lowercases represents to have significant difference, identical lowercase is represented there was no significant difference; α represents utmost point conspicuousness detection level, and different capitalizations represents to have utmost point significant difference, and identical capitalization is represented there was no significant difference.
By table 1 and table 2 as can be known, use pre-treating method provided by the invention, after two reference sample beef were handled, all to measure institute's assignment jointly approaching with European Union 6 isotope analysis laboratories with the accuracy of isotopic mass spectrometry measurement result and the coefficient of variation, there was no significant difference.And after carrying out pre-treatment with the method for bibliographical information and existing national standard (GB/T18932.1-2002) method that provides, use the same method and equipment detect, the carbon isotope result significantly is lower than laboratory institute of European Union assignment (α＜0.01), the nitrogen isotope result is apparently higher than laboratory institute of European Union assignment (α＜0.01), and the coefficient of variation of measuring for 10 times is excessive, shows pre-treating method accurate and effective provided by the invention.
Embodiment 2, the pre-treatment of liquid animal tissue sample
Getting respectively by European Union-food research project of tracing to the source provides, contract number is after each 30ml of two parts of reference sample bovine bloods (being respectively reference sample 1 and reference sample 2) of 006942 thaws slightly, be contained in the pottery in (as small beaker), place refrigerator-80 ℃ after 4-6 hour, to freeze reality, seal film and seal the beaker mouth, the acupuncture aperture, put into freeze drier shown in Figure 2 in-50 ℃ of dryings 24 hours, this moment described reference sample 1 and reference sample 2 bone dry, after taking out described two samples, use the mortar of making by stupalith to grind to crossing 40 purpose aperture sieve, be soaked in again in the methylene chloride in 60 ℃ of degreasings 1 hour, solution after the degreasing is filtered, remove impurity, get filtrate, described filtrate is air-dry under 40 ℃, finish the pre-treatment of described bovine blood sample.
Will with last identical bovine blood sample reference sample 1 and reference sample 2, (this method is: sample is put into 40ml glass centrifuge tube centrifugal (centrifugal force is 15000g), and solid matter filter to be removed in the back to use bibliographical information respectively.Get 10 gram samples and put into the 50ml centrifuge tube, add the 4ml deionized water, the spiral concussion.Get 2-3ml in 80 ℃ of water-baths concussion 3-4 hours at branch, sample centrifugal once more (15000g), get supernatant after, precipitation is with 5ml deionization washing 5 times, precipitation after the dissolving, changes the 1.5ml centrifuge tube over to fully, removes supernatant after centrifugal, and is to be measured after the heat of precipitation drying.) method and the pre-treating method that provided of existing national standard (GB/T18932.1-2002) carry out pre-treatment.And two parts of reference sample bovine bloods (provided by European Union-food research project of tracing to the source, contract number is 006942) are carried out pre-treatment, each sample determination 10 times, each 2 parallel samples.The carbon isotope of these two parts of reference sample bovine bloods and nitrogen isotope are measured assignment jointly by 6 isotope analysis laboratories of European Union.
With the bovine blood sample that three kinds of different pre-treating methods of above-mentioned usefulness dispose, measure the wherein relative abundance of carbon, nitrogen stable isotope according to following method.
1) mensuration program:
Get the 1mg sample, wrap the back with the tinfoil paper cup and deliver to elemental analyser (Flash EA1112 type), treat that carbon and the nitrogen element in the sample is converted into pure CO by automatic sampler
2Gas and N
2Behind the gas, be the dilution instrument of ConfloIII type through model again, enter DELTAPlus Thermo Finnigan mass spectrometer at last and detect.
Concrete running parameter is as follows:
Elemental analyser: the oxidation furnace temperature is 900 ℃, 650 ℃ of reduction furnace temperature, and injector helium purge flow is 300ml/min, oxygen flow is 240ml/min, reference gas flow 200ml/min.
ConfloIII type dilution instrument: helium pressure is 0.6bar, CO
2Reference gas pressure is bar, N
2Reference gas pressure is 1.0bar.
DELTAPlus Thermo Finnigan mass spectrometer: the analytical column temperature is 40 ℃; Demarcate CO with USGS24 (δ 13CPDB=-16.00 ‰)
2Steel cylinder is demarcated N with IAEAN1 (δ 15Nair=0.4 ‰)
2Steel cylinder.With the steel cylinder gas of demarcating as standard.
2) result calculates:
Generally acknowledge in the world and use relative quantity to represent isotopic enrichment degree, computing formula is:
δ ‰=(R sample/R standard-1) * 1000
Wherein, stable carbon, nitrogen isotope ratio are used δ respectively
13C ‰ and δ
15N ‰ expression, R is the abundance ratio of heavy isotope and light isotope in the sample, promptly
13C/
12C and
15N/
14N.International standard substance is the plan belemnite (PDB) of Cretaceous System during South Carolina, United States PeeDee builds to carbon isotope, is atmospheric nitrogen to nitrogen isotope.
3) measurement result:
According to step 2) computing formula, by computer mass spectrometric spectrogram result is calculated according to blas, the measurement result of gained carbon isotope and nitrogen isotope is respectively shown in table 3 and table 4.
The result that table 3, carbon isotope are measured
α
*Expression conspicuousness detection level, different lowercases represents to have significant difference, identical lowercase is represented there was no significant difference; α represents utmost point conspicuousness detection level, and different capitalizations represents to have utmost point significant difference, and identical capitalization is represented there was no significant difference.
The result that table 4, nitrogen isotope are measured
α
*Expression conspicuousness detection level, different lowercases represents to have significant difference, identical lowercase is represented there was no significant difference; α represents utmost point conspicuousness detection level, and different capitalizations represents to have utmost point significant difference, and identical capitalization is represented there was no significant difference.
By table 3 and table 4 as can be known, after with pre-treating method provided by the invention the reference sample bovine blood being handled, all to measure institute's assignment jointly approaching with European Union 6 isotope analysis laboratories with the accuracy of isotopic mass spectrometry measurement result and the coefficient of variation, there was no significant difference.And after carrying out pre-treatment with the method for bibliographical information and existing national standard (GB/T18932.1-2002) method that provides, use the same method and equipment detect, the carbon isotope result significantly is lower than laboratory institute of European Union assignment (α＜0.01), the nitrogen isotope result is apparently higher than laboratory institute of European Union assignment (α＜0.01), and the coefficient of variation of measuring for 10 times is excessive, shows pre-treating method accurate and effective provided by the invention.
Claims (10)
1. a pre-treating method that is used for the animal sample of stable isotope detection comprises the steps:
Animal sample is contained in the utensil that no stable isotope disturbs after the freeze drying, and the utensil grind into powder that disturbs with no stable isotope is finished the described pre-treatment that is used for the animal sample of stable isotope detection again.
2. method according to claim 1 is characterized in that: the utensil of utensil for being made of stupalith that described no stable isotope disturbs.
3. method according to claim 1 and 2 is characterized in that: in the described freeze drying step, temperature is-25 ℃ to-65 ℃.
4. method according to claim 3 is characterized in that: in the described freeze drying step, temperature is-40 ℃.
5. according to the arbitrary described method of claim 1-4, it is characterized in that: the order number of described powder was a 20-40 purpose aperture sieve.
6. method according to claim 5 is characterized in that: the order number of described powder was 40 purpose aperture sieve.
7. according to the arbitrary described method of claim 1-6, it is characterized in that: described animal sample is animal muscle sample or animal body fluid sample.
8. method according to claim 7, it is characterized in that: when described animal sample is the animal body fluid sample, after the step of the described utensil grind into powder that disturbs with no stable isotope, also carry out following processing: described powder is dipped in carries out degreasing in the ethylene dichloride, filter drying.
9. method according to claim 8 is characterized in that: in the described defatting step, the time is 1-2 hour, and preferred 1 hour, temperature was 55-65 ℃, preferred 60 ℃; In the described drying steps, temperature is 25-50 ℃, preferred 40 ℃.
10. according to the arbitrary described method of claim 1-9, it is characterized in that: described animal sample is beef or bovine blood; Described stable isotope is carbon stable isotope and nitrogen stable isotope.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103472124A (en) * | 2013-08-30 | 2013-12-25 | 大连海事大学 | Fingerprint identification method of marine organisms as well as products thereof |
| CN104198216A (en) * | 2014-09-24 | 2014-12-10 | 中国科学院水生生物研究所 | Sampling method of fish body muscle for stable isotope analysis |
| CN106970408A (en) * | 2017-04-11 | 2017-07-21 | 上海海洋大学 | Stable isotope determination pre-treating method in siphonopods spectacle lens |
| CN107741502A (en) * | 2017-10-12 | 2018-02-27 | 中国农业科学院农业质量标准与检测技术研究所 | Carbon, the preparation method of nitrogen standard of stable isotope sample in a kind of beef protein |
| CN108940486A (en) * | 2017-05-26 | 2018-12-07 | 中国农业科学院农业质量标准与检测技术研究所 | Parallel beveller |
| CN110793826A (en) * | 2019-10-31 | 2020-02-14 | 上海化工研究院有限公司 | Sample preparation method for determining carbon 13 isotopic abundance |
-
2010
- 2010-04-26 CN CN 201010155399 patent/CN101813583A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 《中国博士学位论文全文数据库 农业科技辑》 20091015 孙丰梅 应用稳定同位素进行牛肉溯源的研究 24-25 1-10 , 第10期 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103472124A (en) * | 2013-08-30 | 2013-12-25 | 大连海事大学 | Fingerprint identification method of marine organisms as well as products thereof |
| CN104198216A (en) * | 2014-09-24 | 2014-12-10 | 中国科学院水生生物研究所 | Sampling method of fish body muscle for stable isotope analysis |
| CN106970408A (en) * | 2017-04-11 | 2017-07-21 | 上海海洋大学 | Stable isotope determination pre-treating method in siphonopods spectacle lens |
| CN108940486A (en) * | 2017-05-26 | 2018-12-07 | 中国农业科学院农业质量标准与检测技术研究所 | Parallel beveller |
| CN107741502A (en) * | 2017-10-12 | 2018-02-27 | 中国农业科学院农业质量标准与检测技术研究所 | Carbon, the preparation method of nitrogen standard of stable isotope sample in a kind of beef protein |
| CN110793826A (en) * | 2019-10-31 | 2020-02-14 | 上海化工研究院有限公司 | Sample preparation method for determining carbon 13 isotopic abundance |
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Open date: 20100825 |