CN106324147A - Detection method for melamine in milk powder - Google Patents
Detection method for melamine in milk powder Download PDFInfo
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- CN106324147A CN106324147A CN201610942777.9A CN201610942777A CN106324147A CN 106324147 A CN106324147 A CN 106324147A CN 201610942777 A CN201610942777 A CN 201610942777A CN 106324147 A CN106324147 A CN 106324147A
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- milk powder
- tripolycyanamide
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- ammonia
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- 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
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Abstract
The invention discloses a detection method for melamine in milk powder. The method comprises the following steps: pretreating milk powder: weighting 3-5g of dry milk powder, putting into a conical flask cooked and cleaned with acid, adding 50ml of mixed aqueous solution of ammonium hydroxide and ethyl alcohol, sealing the opening with a cover and then putting onto an oscillating instrument; utilizing the oscillating instrument to rotate and oscillate; quickly moving supernate into a centrifugal tube; centrifuging, thereby obtaining a centrifugal liquid which is then put into the clean conical flask; taking a certain quantity of centrifugal liquid, performing rotary evaporation and adopting a buffer organic solvent with pH from 4 to 4.5 for obtaining a constant volume, thereby obtaining an extracting solution; utilizing a solid-phase extracting column to purify the extracting solution, thereby obtaining a purified solution; utilizing high performance liquid chromatography to detect the purified solution, thereby determining the melamine in milk powder. According to the invention, the detection recovery rate can be increased, the times of detecting the recovery rate can be reduced, each sample can reach the standard by only one time of detecting and the problem of component content loss caused by adopting aurilave blowing due to difficulty in column filtration in national standard method is thoroughly solved.
Description
Technical field
The present invention relates to technical field of food detection, the detection method of tripolycyanamide in a kind of milk powder.
Background technology
Sanlu milk powder case, has beaten alarm bell to food safety, and China's dairy industry is caused huge strike.In thing
After part occurs, owing to lacking the means of quickly effectively detection, once creating dairy farmer's fresh milk no one ventured and receiving, have to milk-draining and cow-killing
Vicious cycle.For solving coherent detection problem, " raw milk and melamine in dairy products detection method " has been issued in China's approval
(GB/T 22388-2008), " in raw milk, tripolycyanamide quickly detects liquid chromatography " (GB/T 22400-2008) country mark
Standard and " in outlet liquid milk, tripolycyanamide quickly measures Raman spectroscopy " (SN/T 2805-2011).
The Solid-Phase Extraction pre-treating method that GB/T 22388-2008 provides, cost is high, detection speed is slow, it is difficult to meets and criticizes
Amount detection needs.For method for quick (GB/T 22400-2008), pre-treatment solvent for use acetonitrile is relatively costly,
Needing during operation acutely to shake 6min, the time is the longest, and the individual variation of experimenter, rule of operation implementation status and
The factors such as experimental technique difference of ability all may affect testing result.Raman spectroscopy (SN/T 2805-2011) provided from
Heart pre-treating method, needs configuration high-speed centrifuge instrument, and the operating time is 4min, and pre-treatment gained solution is not clarified, no
Can detect with additive method.
Tripolycyanamide quickly detects for raw milk purchase quality safety significant, although country has issued at present
Cloth quick examination criteria method accordingly, but its in speed, consumables cost, to be prone to the aspect such as universal the most undesirable, therefore enters one
It is the key technical problem being badly in need of at present solving that step improves tripolycyanamide detection method in milk powder.
Summary of the invention
It is an object of the invention to provide the detection method of tripolycyanamide in a kind of milk powder, detection can not only be improved and reclaim
Rate, and the number of times of the detection response rate can be reduced.
For achieving the above object, the present invention provides following technical scheme:
The detection method of tripolycyanamide in a kind of milk powder, comprises the following steps:
(1) milk powder pre-treatment, comprises the following steps:
11) weigh dry milk powder 3～5g, be placed in acid and boil in clean conical flask, add the mixed aqueous solution of ammonia and ethanol
50ml, after lid seal, is placed on shaker；
12) utilize shaker to carry out rotational oscillation, treat that rotational oscillation terminates, quickly supernatant is moved to centrifuge tube, place into from
Scheming is centrifuged, obtains centrifugal liquid, put in clean conical flask, standby；
13) taking out the centrifugal liquid of certain volume, rotary evaporation falls the mixed aqueous solution of ammonia and ethanol, then with pH at 4～4.5 models
Enclose interior buffering organic solvent and carry out constant volume, obtain extracting solution；
(2) with 2ml methanol, 2ml water solid phase extraction column activated the most successively and balance, then extracting solution 10ml is shifted
To solid-phase extraction column；Successively with 2ml water, 2ml methanol drip washing, solid phase extraction column is drained near doing, and uses 5ml ammoniated methanol
Eluant solution；Whole solid phase extraction procedure flow velocity is less than 0.8ml/min；Eluent dries up with nitrogen at 50 DEG C, is dried residual
Stay thing 1ml flowing phase constant volume, whirlpool mixing 1min, cross the organic facies microporous filter membrane of 0.2 μm, be purified liquid；
(3) utilize high performance liquid chromatography detection scavenging solution to measure the tripolycyanamide in milk powder；Wherein, the work of high performance liquid chromatography
As condition it is: chromatographic column is C18 post, 250mm × 4.6mm, 5 μm；Flowing is ion-pairing agent buffer mutually: formic acid=85:15,
Flow velocity is 0.5ml/min, and column temperature is 35 DEG C, and wavelength is 243nm, and sample size is 15 μ L.
As the further scheme of the present invention: in described step 11), in the mixed aqueous solution of ammonia and ethanol, ammonia:
Ethanol: the volume ratio of water is 25～28:55～65:12～15.
As the further scheme of the present invention: in described step 11), in the mixed aqueous solution of ammonia and ethanol, ammonia:
Ethanol: the volume ratio of water is 25:60:15.
As the further scheme of the present invention: in described step 12), rotational oscillation parameter is: speed 120～150r/
Min, the time 15～18min；Parameter of noncentricity is: speed 5500～5800r/min, the time 5～8min.
As the further scheme of the present invention: in described step 12), rotational oscillation parameter is: speed 140r/min, time
Between 16min；Parameter of noncentricity is: speed 5600r/min, time 7min.
As the further scheme of the present invention: in described step 13), with the salt buffer of 2% acetum that pH is 4.2
Solution carrys out constant volume.
As the further scheme of the present invention: in described step (3), the response rate of tripolycyanamide=(spiked levels
Actually detected result-blank result)/theoretical spiked levels × 100%, wherein, theoretical spiked levels refers to that sample is untreated front additional
To the content of the tripolycyanamide of this testing sample, the actually detected result of spiked levels refers to the outer melamine adding to this testing sample
The content of amine result after sample treatment and chromatograph detect, blank result is in this testing sample of the most additional tripolycyanamide
The testing result of the content of tripolycyanamide.
Compared with prior art, the invention has the beneficial effects as follows: the present invention uses mechanical oscillation, alkaline organic compound to enter
Row extracts, and uses pH buffering organic solvent in the range of 4～4.5 to be diluted constant volume as diluent, then is extracted by solid phase
Take post to purify, finally utilize high performance liquid chromatography to detect the tripolycyanamide obtaining in milk powder；The method can not only improve
The detection response rate, and the number of times of the detection response rate can be reduced so that every sample has only to detect once just can be up to standard, thoroughly solves
National Standard Method of having determined use ear washing bulb blow caused constituent content loss problem because of post filtration difficulty.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, all
Belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, the detection method of tripolycyanamide in a kind of milk powder, comprise the following steps:
(1) milk powder pre-treatment, comprises the following steps:
11) weigh dry milk powder 3～5g, be placed in acid and boil in clean conical flask, add the mixed aqueous solution of ammonia and ethanol
(ammonia: ethanol: the volume ratio of water is 25:60:15) 50ml, after lid seal, is placed on shaker；
12) utilize shaker to carry out rotational oscillation, treat that rotational oscillation terminates, quickly supernatant is moved to centrifuge tube, place into from
Scheming is centrifuged, obtains centrifugal liquid, put in clean conical flask, standby；Wherein, rotational oscillation parameter is: speed 140r/
Min, time 16min；Parameter of noncentricity is: speed 5600r/min, time 7min；
13) taking out centrifugal liquid 5ml, rotary evaporation falls the mixed aqueous solution of ammonia and ethanol, then with pH in the range of 4～4.5
Buffering organic solvent, preferably pH be the salt buffer solution of 2% acetum of 4.2 to be settled to 5ml, obtain extracting solution；
(2) with 2ml methanol, 2ml water solid phase extraction column activated the most successively and balance, then extracting solution 10ml is shifted
To solid-phase extraction column；Successively with 2ml water, 2ml methanol drip washing, solid phase extraction column is drained near doing, and uses 5ml ammoniated methanol
Eluant solution；Whole solid phase extraction procedure flow velocity is less than 0.8ml/min；Eluent dries up with nitrogen at 50 DEG C, is dried residual
Stay thing 1ml flowing phase constant volume, whirlpool mixing 1min, cross the organic facies microporous filter membrane of 0.2 μm, be purified liquid；
(3) utilize high performance liquid chromatography detection scavenging solution to measure the tripolycyanamide in milk powder；Wherein, the work of high performance liquid chromatography
As condition it is: chromatographic column is C18 post, 250mm × 4.6mm, 5 μm；Flowing is ion-pairing agent buffer mutually: formic acid=85:15,
Flow velocity is 0.5ml/min, and column temperature is 35 DEG C, and wavelength is 243nm, and sample size is 15 μ L.
The response rate=(the actually detected result-blank result of spiked levels)/theory spiked levels × 100%, wherein, reason
Opinion spiked levels refers to the content of the untreated front outer tripolycyanamide adding to this testing sample of sample, the actually detected knot of spiked levels
Really referring to the content adding to outward the tripolycyanamide of this testing sample result after sample treatment and chromatograph detect, blank result is not
The testing result of the content of the tripolycyanamide in this testing sample of additional tripolycyanamide.
Utilize national standard method, the inventive method both approaches, to milk powder (A), additional melamine concentration be respectively
This milk powder (B) of 2ppm, additional melamine concentration are that this milk powder (C) of 4ppm carries out melamine concentration detection, and calculating should
The tripolycyanamide detection response rate of milk powder, often organizes in triplicate, and testing result takes three meansigma methodss, takes 2 Duplicate Samples every time.Right
More as shown in table 1 than result.
The result of the test of 1 two kinds of methods of table compares
As can be seen from Table 1: one, the inventive method can improve the detection response rate, specifically can be by about the 70% of National Standard Method
The response rate brings up to the response rate of more than 83%.Such as, utilize National Standard Method, post filtration difficulty, need to use ear washing bulb to blow solid phase extraction
Taking the scavenging solution in post, during blowing, the component to be measured in scavenging solution will be carried over, because using ear washing bulb to blow
During it cannot be guaranteed that the speed of 1/4 seconds, the content of component the most to be measured is lost；Utilize the inventive method,
Post filters smooth and easy, it is not necessary to use ear washing bulb to blow the scavenging solution in solid-phase extraction column pillar, makes extracting solution naturally flow out solid phase extraction
Taking pillar, the component to be measured in scavenging solution naturally also can be adsorbed in solid phase extraction column, the loss of the content in component to be measured
Relatively reduce.Two, the inventive method can reduce the number of times of the detection response rate.Such as, before improving, every sample detection response rate is secondary
Number typically will can be only achieved the prescribed limit of the response rate for 2～3 times；And utilize the every sample of the inventive method to have only to the most just detect
Can be up to standard.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of the spirit or essential attributes of the present invention, it is possible to realize the present invention in other specific forms.Therefore, no matter
From the point of view of which point, all should regard embodiment as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all by fall in the implication of equivalency and scope of claim
Change is included in the present invention.
Although moreover, it will be appreciated that this specification is been described by according to embodiment, but the most each embodiment only wraps
Containing an independent technical scheme, this narrating mode of description is only that for clarity sake those skilled in the art should
Description can also be formed those skilled in the art through appropriately combined as an entirety, the technical scheme in each embodiment
May be appreciated other embodiments.
Claims (7)
1. the detection method of tripolycyanamide in a milk powder, it is characterised in that comprise the following steps:
(1) milk powder pre-treatment, comprises the following steps:
11) weigh dry milk powder 3～5g, be placed in acid and boil in clean conical flask, add the mixed aqueous solution of ammonia and ethanol
50ml, after lid seal, is placed on shaker；
12) utilize shaker to carry out rotational oscillation, treat that rotational oscillation terminates, quickly supernatant is moved to centrifuge tube, place into from
Scheming is centrifuged, obtains centrifugal liquid, put in clean conical flask, standby；
13) taking out the centrifugal liquid of certain volume, rotary evaporation falls the mixed aqueous solution of ammonia and ethanol, then with pH at 4～4.5 models
Enclose interior buffering organic solvent and carry out constant volume, obtain extracting solution；
(2) with 2ml methanol, 2ml water solid phase extraction column activated the most successively and balance, then extracting solution 10ml is shifted
To solid-phase extraction column；Successively with 2ml water, 2ml methanol drip washing, solid phase extraction column is drained near doing, and uses 5ml ammoniated methanol
Eluant solution；Whole solid phase extraction procedure flow velocity is less than 0.8ml/min；Eluent dries up with nitrogen at 50 DEG C, is dried residual
Stay thing 1ml flowing phase constant volume, whirlpool mixing 1min, cross the organic facies microporous filter membrane of 0.2 μm, be purified liquid；
(3) utilize high performance liquid chromatography detection scavenging solution to measure the tripolycyanamide in milk powder；Wherein, the work of high performance liquid chromatography
As condition it is: chromatographic column is C18 post, 250mm × 4.6mm, 5 μm；Flowing is ion-pairing agent buffer mutually: formic acid=85:15,
Flow velocity is 0.5ml/min, and column temperature is 35 DEG C, and wavelength is 243nm, and sample size is 15 μ L.
The detection method of tripolycyanamide in milk powder the most according to claim 1, it is characterised in that in described step 11),
In the mixed aqueous solution of ammonia and ethanol, ammonia: ethanol: the volume ratio of water is 25～28:55～65:12～15.
The detection method of tripolycyanamide in milk powder the most according to claim 2, it is characterised in that in described step 11),
In the mixed aqueous solution of ammonia and ethanol, ammonia: ethanol: the volume ratio of water is 25:60:15.
The detection method of tripolycyanamide in milk powder the most according to claim 1, it is characterised in that in described step 12),
Rotational oscillation parameter is: speed 120～150r/min, the time 15～18min；Parameter of noncentricity is: speed 5500～5800r/
Min, the time 5～8min.
The detection method of tripolycyanamide in milk powder the most according to claim 4, it is characterised in that in described step 12),
Rotational oscillation parameter is: speed 140r/min, time 16min；Parameter of noncentricity is: speed 5600r/min, time 7min.
The detection method of tripolycyanamide in milk powder the most according to claim 1, it is characterised in that in described step 13),
Constant volume is carried out with the salt buffer solution of 2% acetum that pH is 4.2.
The detection method of tripolycyanamide in milk powder the most according to claim 1, it is characterised in that in described step (3),
The response rate of tripolycyanamide=(the actually detected result-blank result of spiked levels)/theory spiked levels × 100%, wherein,
Theoretical spiked levels refer to sample untreated before the content of the outer tripolycyanamide adding to this testing sample, spiked levels actually detected
Result refers to the content of the outer tripolycyanamide adding to this testing sample result after sample treatment and chromatograph detect, and blank result is
The testing result of the content of the tripolycyanamide in this testing sample of the most additional tripolycyanamide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610942777.9A CN106324147A (en) | 2016-11-02 | 2016-11-02 | Detection method for melamine in milk powder |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610942777.9A CN106324147A (en) | 2016-11-02 | 2016-11-02 | Detection method for melamine in milk powder |
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| CN106324147A true CN106324147A (en) | 2017-01-11 |
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| CN201610942777.9A Pending CN106324147A (en) | 2016-11-02 | 2016-11-02 | Detection method for melamine in milk powder |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108802236A (en) * | 2018-07-10 | 2018-11-13 | 南方医科大学 | A method of measuring melamine in dairy products content |
| CN109239049A (en) * | 2018-09-13 | 2019-01-18 | 常州常检诺食品检测中心有限公司 | The detection method of melamine in a kind of milk powder |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0390858A (en) * | 1989-09-01 | 1991-04-16 | Shimadzu Corp | Analysis of molecular weight distribution of melanin |
| CN101513577A (en) * | 2009-03-01 | 2009-08-26 | 国家海洋局第一海洋研究所 | Melamine molecular imprinted solid phase extraction cartridge and a preparation method and application thereof |
| CN101968452A (en) * | 2010-03-05 | 2011-02-09 | 浙江省疾病预防控制中心 | Kit for quickly detecting tripolycyanamide and use thereof |
| CN102879504A (en) * | 2012-10-22 | 2013-01-16 | 天津出入境检验检疫局工业产品安全技术中心 | Melamine detection method |
| US20140036263A1 (en) * | 2012-07-31 | 2014-02-06 | Ansoon Kim | Apparatus for performing spectroscopy having a porous membrane |
-
2016
- 2016-11-02 CN CN201610942777.9A patent/CN106324147A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0390858A (en) * | 1989-09-01 | 1991-04-16 | Shimadzu Corp | Analysis of molecular weight distribution of melanin |
| CN101513577A (en) * | 2009-03-01 | 2009-08-26 | 国家海洋局第一海洋研究所 | Melamine molecular imprinted solid phase extraction cartridge and a preparation method and application thereof |
| CN101968452A (en) * | 2010-03-05 | 2011-02-09 | 浙江省疾病预防控制中心 | Kit for quickly detecting tripolycyanamide and use thereof |
| US20140036263A1 (en) * | 2012-07-31 | 2014-02-06 | Ansoon Kim | Apparatus for performing spectroscopy having a porous membrane |
| CN102879504A (en) * | 2012-10-22 | 2013-01-16 | 天津出入境检验检疫局工业产品安全技术中心 | Melamine detection method |
Non-Patent Citations (3)
| Title |
|---|
| 乔勇升 等: "高效液相色谱对液态奶中三聚氰胺的快速测定", 《分析测试学报》 * |
| 孙明星 等: "土壤中三聚氰胺的高效液相色谱分析", 《上海交通大学学报（农业科学版）》 * |
| 饶钦雄 等: "饲料和食品中三聚氰胺的毒性及残留检测方法的研究进展", 《饲料工业》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108802236A (en) * | 2018-07-10 | 2018-11-13 | 南方医科大学 | A method of measuring melamine in dairy products content |
| CN108802236B (en) * | 2018-07-10 | 2021-05-18 | 南方医科大学 | Method for determining melamine content in dairy product |
| CN109239049A (en) * | 2018-09-13 | 2019-01-18 | 常州常检诺食品检测中心有限公司 | The detection method of melamine in a kind of milk powder |
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