CN105548433B - The method that liquid chromatography tandem mass spectrometry detects various non-protein nitrogen-containing compound contents in milk simultaneously - Google Patents
The method that liquid chromatography tandem mass spectrometry detects various non-protein nitrogen-containing compound contents in milk simultaneously Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 64
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 64
- -1 nitrogen-containing compound Chemical class 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 27
- 235000013336 milk Nutrition 0.000 title claims abstract description 22
- 239000008267 milk Substances 0.000 title claims abstract description 22
- 210000004080 milk Anatomy 0.000 title claims abstract description 22
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 136
- 239000007788 liquid Substances 0.000 claims abstract description 53
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 40
- 229920000877 Melamine resin Polymers 0.000 claims abstract description 29
- PKTIFYGCWCQRSX-UHFFFAOYSA-N 4,6-diamino-2-(cyclopropylamino)pyrimidine-5-carbonitrile Chemical compound NC1=C(C#N)C(N)=NC(NC2CC2)=N1 PKTIFYGCWCQRSX-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000005891 Cyromazine Substances 0.000 claims abstract description 22
- LVQDKIWDGQRHTE-UHFFFAOYSA-N cyromazine Chemical compound NC1=NC(N)=NC(NC2CC2)=N1 LVQDKIWDGQRHTE-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229950000775 cyromazine Drugs 0.000 claims abstract description 22
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 claims abstract description 22
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 20
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 20
- 235000019253 formic acid Nutrition 0.000 claims abstract description 20
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims abstract description 19
- 235000019257 ammonium acetate Nutrition 0.000 claims abstract description 19
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 claims abstract description 19
- 238000012360 testing method Methods 0.000 claims abstract description 19
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 13
- 239000012224 working solution Substances 0.000 claims abstract description 13
- 239000003480 eluent Substances 0.000 claims abstract description 10
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 5
- 239000004519 grease Substances 0.000 claims abstract description 5
- 238000001914 filtration Methods 0.000 claims abstract description 3
- 239000007800 oxidant agent Substances 0.000 claims abstract description 3
- 230000001590 oxidative effect Effects 0.000 claims abstract description 3
- 150000002500 ions Chemical class 0.000 claims description 36
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 32
- 150000001875 compounds Chemical class 0.000 claims description 32
- JDSHMPZPIAZGSV-UHFFFAOYSA-O melamine(1+) Chemical compound NC1=NC(N)=[NH+]C(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-O 0.000 claims description 27
- 239000012071 phase Substances 0.000 claims description 27
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 19
- 229910052757 nitrogen Inorganic materials 0.000 claims description 16
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 14
- 238000004949 mass spectrometry Methods 0.000 claims description 10
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- JDSHMPZPIAZGSV-DPZTXFNWSA-N 1,3,5-triazine-2,4,6-triamine Chemical compound NC1=NC(N)=[15N]C([15NH2])=[15N]1 JDSHMPZPIAZGSV-DPZTXFNWSA-N 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000000889 atomisation Methods 0.000 claims description 2
- 238000002013 hydrophilic interaction chromatography Methods 0.000 claims description 2
- 239000007791 liquid phase Substances 0.000 claims description 2
- 239000012982 microporous membrane Substances 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims description 2
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- 239000005695 Ammonium acetate Substances 0.000 abstract description 14
- 229940043376 ammonium acetate Drugs 0.000 abstract description 14
- 230000035945 sensitivity Effects 0.000 abstract description 4
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 37
- 238000011084 recovery Methods 0.000 description 32
- 239000000243 solution Substances 0.000 description 28
- 230000000052 comparative effect Effects 0.000 description 23
- 230000003595 spectral effect Effects 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 238000000746 purification Methods 0.000 description 12
- 235000013365 dairy product Nutrition 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000011159 matrix material Substances 0.000 description 5
- 150000001412 amines Chemical class 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 4
- 235000020183 skimmed milk Nutrition 0.000 description 4
- 235000008939 whole milk Nutrition 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002203 pretreatment Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000021393 food security Nutrition 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000019750 Crude protein Nutrition 0.000 description 1
- XZMCDFZZKTWFGF-UHFFFAOYSA-N Cyanamide Chemical compound NC#N XZMCDFZZKTWFGF-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- SOSWVVMGPYTPKV-UHFFFAOYSA-N acetic acid;azane;formic acid Chemical compound [NH4+].OC=O.CC([O-])=O SOSWVVMGPYTPKV-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000010813 internal standard method Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000002608 ionic liquid Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 235000014666 liquid concentrate Nutrition 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000007974 melamines Chemical class 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000013613 poultry product Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000000348 solid-phase epitaxy Methods 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000005829 trimerization reaction Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
Classifications
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
A kind of method for detecting various non-protein nitrogen-containing compound contents in milk simultaneously the invention discloses liquid chromatography tandem mass spectrometry, the method includes：To protein precipitant is added in testing sample, it is vortexed, centrifugation, to grease removal agent is added in supernatant, is vortexed, centrifugation, reject fat oxidant layer is dissolved with ammoniacal liquor acetonitrile after lower clear liquid is concentrated, is vortexed, and crosses HLB solid-phase extraction columns, collects eluent, concentration is 1 with volume ratio:9 0.15% aqueous formic acid (ammonium acetate containing 5mM) acetonitrile mixture dissolving, filtering obtains prepare liquid；Preparation standard hybrid working solution series；Standard hybrid working solution series and prepare liquid are distinguished into sample introduction under the conditions of Liquid Chromatography-Tandem Mass Spectrometry, standard curve is made, according to corresponding non-protein nitrogen-containing compound content in standard curve calculating prepare liquid.The present invention can be used to detect simultaneously in Dicyclanil, dicyandiamide, biuret, cyromazine and melamine at least two content, and the degree of accuracy is high, and sensitivity is strong.
Description
Technical field
The present invention relates to a kind of detection method of non-protein nitrogen-containing compound content in dairy products, and in particular to liquid chromatogram
The method that tandem mass spectrometry detects various non-protein nitrogen-containing compound contents in milk simultaneously.
Background technology
Dairy products are one of most ancient natural drink of the mankind, because its is nutritious, easily digest and assimilate, thing U.S. valency
Honest and clean, instant, therefore also referred to as " white blood ".That is most favored in dairy products surely belongs to milk and milk product.
Improved constantly along with the degree of recognition to nutritional value of milk, people also constantly increase to its consumption demand, to its quality requirement
Also more and more higher.
Milk nitrogen content is always an important indicator for judging milk quality, the measure side with Kjeldahl's method as representative
Method can realize the measure to crude protein content in sample.But the method can only measure nitrogen content and cannot be distinguished by the source of nitrogen,
So that nonprotein nitrogen is adulterated cannot be effectively controlled, substandard product is particularly adulterated dairy products serious harm human body and is good for
Health, wherein being protruded the most with the melamine event of Sanlu, the permanent natural also inspection in its product of New Zealand dairy products giant in 2012
Survey and find there is dicyandiamide residual in the dairy produce of part.
The non-protein nitrogen-containing compound that has now been found that generally have it is relatively tasteless, it is colourless and the features such as have a nasty taste,
Food producer deliberately will not generally be carried out using highly toxic compound it is adulterated, however, such as melamine event, even
The chemical substance of relative nontoxic is likely to that unpredictalbe negative effect that cause health and cannot retrieve can be caused.Therefore some
Countries and regions have carried out clear stipulaties to the service condition of non-protein nitrogen-containing compound in food：USA and EU specifies livestock products
Cyromazine highest residual quantity is 0.05mg/kg, the Ministry of Agriculture of China in 2002 in product《Animal food veterinary drug residue highest is limited
Amount》Middle regulation cyromazine MRL in poultry product is 0.05mg/kg, in sheep muscle, fat, liver, kidney most
High residue limitation 0.3mg/kg.The limitation requirement of regulation melamine in dairy products in 2011：Milk power for infant and young children 1mg/kg,
Liquid milk (including raw milk), milk powder, other prescription emulsifiable powders 2.5mg/kg, other food 2.5mg/kg containing breast more than 15%.
After melamine event and dicyandiamide are found, researcher is to its melamine, dicyandiamide and the like
Assay method launch series research, develop use AAS respectively, infra-red sepectrometry, gas chromatography-mass spectrography,
The detection technique of high performance liquid chromatography and Liquid Chromatography-Tandem Mass Spectrometry is measured to it, and China formulates series detection mark
Standard is (for example：GB 29704-2013, GB/T 22388-2008, SN/T 3032-2011), realize the standard to its detection technique
Change.But the involved compound numbers of these detections are relatively single.
With the raising of people's Consciousness of food security, according to traditional analysis method and thinking, it is necessary to detection week more long
Phase, more man power and material, testing cost is high, it is impossible to meets current classes of compounds and is continuously increased, analysis throughput is constantly carried
Demand high.Situation generation (such as trimerization for launching detection is passively tackled again after also easily occurring in generation accident simultaneously
Cyanamide event).Therefore the detection range of residual need from single detection develop into multiple compounds while qualitative and quantitative point
Analysis.
The content of the invention
Detect various non-protein nitrogen-containing compounds in milk simultaneously the invention provides a kind of liquid chromatography tandem mass spectrometry
The method of content, this method solve the prior art problem single to non-protein nitrogen-containing compound detection number.
The method that liquid chromatography tandem mass spectrometry detects various non-protein nitrogen-containing compound contents in milk simultaneously, including with
Lower step：
(1) to protein precipitant is added in testing sample, it is vortexed, centrifugation takes supernatant；
(2) to grease removal agent is added in the supernatant, it is vortexed, centrifugation, reject fat oxidant layer uses ammoniacal liquor after lower clear liquid is concentrated
Acetonitrile dissolving, vortex, obtain liquid to be clean；
(3) liquid to be clean is crossed into HLB solid-phase extraction columns, it is 1 to collect eluent and volume ratio is used after being concentrated:9
The dissolving of 0.15% aqueous formic acid-acetonitrile mixture, constant volume, filtering with microporous membrane obtains prepare liquid；
Contain 3~10mM ammonium acetates in 0.15% aqueous formic acid；
(4) prepare and contain containing Dicyclanil, dicyandiamide, biuret, cyromazine and the non-protein of melamine at least two
The standard mixed solution of nitrogen compound, gradient dilution is carried out by standard mixed solution, obtains standard hybrid working solution series；
(5) by the serial and described prepare liquid of the standard hybrid working solution in default Liquid Chromatography-Tandem Mass Spectrometry condition
Lower sample introduction respectively, the testing result according to standard hybrid working solution series makes standard curve, further according to standard curve and treating
The testing result for surveying liquid calculates the content of corresponding non-protein nitrogen-containing compound in prepare liquid；
Wherein, liquid phase chromatogram condition is：
Chromatographic column：Waters XBridge HILIC, 4.6mm × 150mm, 3.5 μm；
Mobile phase A：0.15% aqueous formic acid, containing 3~10mM ammonium acetates；
Mobile phase B：Acetonitrile；
Flow velocity：0.4mL/min；
Sample size：10μL；
Column temperature：40℃；
Gradient elution program：①0min：10%A, 90%B；
②3.0min：50%A, 50%B；
③8.0min：50%A, 50%B；
④9.0min：10%A, 90%B；
⑤20.0min：10%A, 90%B；
Mass Spectrometry Conditions are：
Ion gun：Electric spray ion source；
Scan mode：Cation is scanned；
Monitoring mode：Multiple-reaction monitoring；
Electron spray voltage：3500V；
Ion source temperature：250℃；
Dry temperature degree：High pure nitrogen, 250 DEG C；
Dry gas stream amount：High pure nitrogen, 16L/min；
Atomization gas pressure：High pure nitrogen, 275.8kPa；
Sheath temperature degree：High pure nitrogen, 350 DEG C；
Sheath throughput：High pure nitrogen, 10L/min；
Mass spectrometry parameters are：
6. Dicyclanil：Quota ion pair is 191.1/150.0, and collision energy is 21；Qualitative ion pair is 191.1/
109.1, collision energy is 31；
7. dicyandiamide：Quota ion pair is 85.1/68.1, and collision energy is 20；Qualitative ion pair is 85.1/43.1, is touched
It is 22 to hit energy；
8. biuret：Quota ion pair is 104.2/61.0, and collision energy is 9；Qualitative ion pair is 104.2/44.1, is touched
It is 37 to hit energy；
9. cyromazine：Quota ion pair is 167.2/85.1, and collision energy is 20；Qualitative ion pair is 167.2/
125.1, collision energy is 18；
10. melamine：Quota ion pair is 127.1/85.1, and collision energy is 19；Qualitative ion pair is 127.1/
68.1, collision energy is 36.
The present invention can be used to detecting in Dicyclanil, dicyandiamide, biuret, cyromazine and melamine that at least two is non-
The content of albumen nitrogen-containing compound, and be equal in terms of 10 by S/N, the measure to each non-protein nitrogen-containing compound in milk sample is determined
Amount limit is reachable：The μ g/kg of Dicyclanil 0.5, the μ g/kg of dicyandiamide 2.5, the μ g/kg of biuret 20, the μ g/kg of cyromazine 5, melamine
The μ g/kg of amine 15, the degree of accuracy is high, and sensitivity is strong.
After the present invention is through albumen precipitation, degreasing, HLB SPEs are coordinated to be purified using ammoniacal liquor acetonitrile.With acetonitrile phase
Than elution efficiency of the liquid to be clean on HLB solid-phase extraction columns can not only be improved using ammoniacal liquor acetonitrile, moreover it is possible to improves the rate of recovery.
And with QuEChERS purification techniques and other solid-phase extraction columns (such as MCX, C18) compare, carried out only using HLB solid-phase extraction columns
During change, more than 80% can be reached to five kinds of rate of recovery of non-protein nitrogen-containing compound.
The present invention (contains 3~10mM in integrated survey acetonitrile, methyl alcohol, 0.15% aqueous formic acid, 0.15% aqueous formic acid
Ammonium acetate) for constant volume solution and mobile phase when separation, spectral peak peak shape and intensity when find, mobile phase be acetonitrile and
During 0.15% aqueous formic acid (gradient elution), the peak shape and intensity of spectral peak preferably, further can be carried substantially after adding ammonium acetate
The signal intensity (wherein the signal intensity of Dicyclanil improves nearly 50 times) of each compound high.Research discovery, Dicyclanil spectral peak
Peak shape is influenceed larger by extraneous factor, when constant volume solution is consistent with mobile phase initial soln, Dicyclanil spectral peak peak shape
Preferably, the two is inconsistent, and " bifurcated " peak occurs.Therefore the constant volume solution and mobile phase initial soln phase for being used in step (3)
Unanimously.
As further preferred, the melamine-15N3The addition of internal standard compound is 50~200ng；More preferably
100ng。
As further preferred, the melamine-15N3The mass spectrometry parameters of internal standard compound are：Qualitative ion pair is 130.2/
87.1, collision energy is 19.
Preferably, in step (1), the protein precipitant is acetonitrile.Compared with conventional trichloroacetic acid and methyl alcohol, adopt
During with acetonitrile as protein precipitant, both it had been easy to carry out follow-up lower clear liquid rapid concentration, had improved the signal of target detection thing
Responsiveness, can realize more thoroughly precipitating, it is to avoid protein residues influence detection knot to the protein in testing sample again
Really.
Used as further preferred, acetonitrile is (15~20) with the mixed volume ratio of testing sample：1.
Preferably, in step (2), the grease removal agent is n-hexane.During vortex, a large amount of lipids can be extracted into
In n-hexane layer, while the rate of recovery is not influenceed, the interference of impurity background is greatly reduced.
Ungrease treatment is carried out using the n-hexane of acetonitrile saturation, the detection rate of recovery can be further improved.
Used as further preferred, n-hexane is (1~1.5) with the mixed volume ratio of the supernatant：2.
Preferably, in step (2), being dissolved with 3~10% ammoniacal liquor acetonitriles after the lower clear liquid is concentrated；More preferably
5% ammoniacal liquor acetonitrile.Using the ammoniacal liquor acetonitrile solution dissolving of above-mentioned volume fraction when the HLB solid-phase extraction columns, it is possible to achieve complete
Full wash-out, and alkalescence will not be too strong, both avoids influenceing the stability of each compound, also saves reagent.
Preferably, in step (3), the concentration of ammonium acetate is 5mM in 0.15% aqueous formic acid.The end of ammonium acetate
When concentration is 5mM, the signal intensity of each compound is maximum.
Preferably, in step (3), the aperture of the miillpore filter is 0.22 μm.
It is general first to adding appropriate ammoniacal liquor acetonitrile to be activated in HLB solid-phase extraction columns before step (3) crosses post, make
Ammoniacal liquor acetonitrile is identical with the ammoniacal liquor acetonitrile of dissolving lower clear liquid concentrate.
Preferably, in step (3), the liquid to be clean is crossed into HLB solid-phase extraction columns, an eluent is collected；Again to
The wash-out solution (i.e. 5% ammoniacal liquor acetonitrile) isometric with liquid to be clean is added in the container for containing liquid to be clean, washed, crossed HLB
Solid-phase extraction column, collects secondary eluent；Merge an eluent and secondary eluent, concentration.So avoid container inner wall residual
The liquid to be clean for staying influences final accuracy in detection and the rate of recovery.
Compared with prior art, beneficial effects of the present invention are：
(1) detection method of the invention can be used to detect Dicyclanil, dicyandiamide, biuret, cyromazine and melamine
The content of at least two non-protein nitrogen-containing compounds in amine, and be equal in terms of 10 by S/N, each non-protein nitrogen in milk sample
The measure quantitative limit of compound is reachable：The μ g/kg of Dicyclanil 0.5, the μ g/kg of dicyandiamide 2.5, biuret 20 μ g/kg, the μ of cyromazine 5
G/kg, the μ g/kg of melamine 15, the degree of accuracy is high, and sensitivity is strong.
(2) present invention coordinates HLB solid-phase extraction columns to be purified using ammoniacal liquor acetonitrile, can not only be improved with ammoniacal liquor acetonitrile and treated
Elution efficiency of the scavenging solution on HLB solid-phase extraction columns, moreover it is possible to improve the rate of recovery, and HLB solid-phase extraction columns are to five kinds of non-protein
The rate of recovery of nitrogen-containing compound can reach more than 80%；
(3) present invention uses LC-MS/MS, linear to close using internal standard and external standard dilution standard measure
System is good, and coefficient correlation is more than 0.995；The overall rate of recovery reaches 78.5%~101.8%；Relative standard deviation 1.3%~
12.3%, accuracy in detection is high, and reappearance, practicality are good；Using internal standard method and external standard method simultaneous quantitative, inspection is further increased
The accuracy rate of survey method；
(4) present invention as mobile phase, is obtained using acetonitrile and 0.15% aqueous formic acid (ammonium acetate containing 3~10mM)
Spectral peak peak shape and signal intensity preferably, can further significantly improve the signal intensity of each compound (wherein after addition ammonium acetate
The signal intensity of former times Neil improves nearly 50 times)；
(5) the constant volume solution that the present invention is used is consistent with mobile phase initial soln, so avoids Dicyclanil spectral peak peak
There is " bifurcated " peak in shape；
(6) detection method of the invention is simple and efficient, and consumption resource is small, and testing cost is low, mentioned pretreatment process,
The organic solvent and measure instrument of use can carry out good complementation with existing detection method, it is adaptable to dairy products
In various non-protein nitrogen-containing compounds carry out the requirement that determines simultaneously, can be to safeguard food security and ensure China dairy products matter
Amount provides strong technical guarantee.
Brief description of the drawings
Fig. 1 is five kinds of MRM total ion current figures of non-protein nitrogen-containing compound；
Wherein, intensity/cps represents the response intensity (cps) of signal, and T/min represents retention time (minute), peak
1. 2. 3. 4. it is that 5. cyromazine, peak are melamine for biuret, peak for dicyandiamide, peak for Dicyclanil, peak；
Fig. 2 is that to use volume ratio be 1:9 0.15% aqueous formic acid (ammonium acetate containing 5mM)-acetonitrile mixture is used as fixed
Hold separation appearance, spectral peak peak shape and the signal intensity of each compound in the matrix solution prepared when solution and mobile phase initial soln
Situation；
Fig. 3 is that to use volume ratio be 1:9 0.15% aqueous formic acid-acetonitrile mixture is used as constant volume solution and mobile phase
The separation appearance of each compound, spectral peak peak shape and signal strength condition in the matrix solution prepared during initial soln；
Fig. 4 is that to use volume ratio be 1:9 0.15% aqueous formic acid-acetonitrile mixture is as constant volume solution, using body
Product is than being 1:Prepared when 9 0.15% aqueous formic acid (ammonium acetate containing 5mM)-acetonitrile mixture is as mobile phase initial soln
The separation appearance of each compound, spectral peak peak shape and signal strength condition in matrix solution；
Wherein, abscissa " Counts vs.Acquisition time (min) " represents retention time (minute), ordinate
Represent signal intensity.
Specific embodiment
Technical scheme is described in further detail with reference to the accompanying drawings and detailed description.
The essential information of five kinds of non-protein nitrogen-containing compounds is as shown in table 1 in the present invention：
1 five kinds of essential informations of non-protein nitrogen-containing compound of table
Embodiment 1
The method that liquid chromatography tandem mass spectrometry detects various non-protein nitrogen-containing compound contents in milk simultaneously, including with
Lower step：
(1) testing sample pre-treatment：
Suck up milk sample 1.00g (being accurate to 0.01g) adds 100ng melamines in 50mL tool plug centrifuge tubes
-15N3Internal standard compound, adds acetonitrile to 20mL, is vortexed, ultrasound, and high speed centrifugation takes supernatant 10mL, adds 7mL n-hexanes, whirlpool
Rotation, removes upper strata n-hexane phase, lower clear liquid be concentrated into it is near dry after add ammoniacal liquor acetonitrile solution that 3mL volume fractions are 5% (below
Referred to as 5% ammoniacal liquor acetonitrile) dissolved, be vortexed, obtain liquid to be clean；
The 5% ammoniacal liquor acetonitrile for adding 5mL to HLB solid-phase extraction columns is activated, and liquid to be clean is crossed into HLB solid phases after activation
Extraction column, collects an eluent, then to adding the wash-out solution isometric with liquid to be clean in the container for containing liquid to be clean
(i.e. 5% ammoniacal liquor acetonitrile), washs, crosses HLB solid-phase extraction columns, collects secondary eluent；Merge an eluent and secondary wash-out
Liquid, is concentrated under reduced pressure into and closely does, and is 1 with volume ratio:9 0.15% aqueous formic acid (ammonium acetate containing 5mM)-acetonitrile mixture dissolving
2mL is settled to, 0.22 μm of miillpore filter is crossed, prepare liquid is obtained；
(2) standard hybrid working solution series is prepared：
Dicyclanil, dicyandiamide, biuret, cyromazine and melamine standard items 10mg are first weighed respectively, use acetonitrile
Dissolving respectively is settled to 10mL, obtains the standard reserving solution of 1.0mg/mL；
Pipette the μ L of each standard reserving solution 100 respectively again, 10mL is settled to acetonitrile, the standard mixing for being made 10 μ g/mL is molten
Liquid；
It is finally 1 with volume ratio:9 that 0.15% aqueous formic acid (ammonium acetate containing 5mM)-acetonitrile mixture carries out gradient is dilute
Release, it is standard hybrid working stock solution series to obtain 0ng/mL, 62.5ng/mL, 125ng/mL, 250ng/mL, 500ng/mL；
Take do not contain Dicyclanil, dicyandiamide, biuret, cyromazine and melamine blank skim milk it is (or complete
Fat milk), processed using step (1) and (2), obtain vehicle solution；
Standard hybrid working stock solution each 0.1mL of series is taken, each to add 0.4mL vehicle solutions, obtaining concentration is
0,12.5,25.0,50.0,100ng/mL (respective quality concentration be 0 μ g/kg, 50 μ g/kg, 100 μ g/kg, 200 μ g/kg, 400 μ
G/kg standard hybrid working solution series) (for being used under cation drainage pattern)；
(3) standard curve is prepared：
Standard hybrid working solution series is distinguished into sample introduction under the conditions of default Liquid Chromatography-Tandem Mass Spectrometry, it is dense with quality
Degree X (μ g/kg) is abscissa, and the ratio Y of peak area is ordinate, draws 5 standard working curves (such as table 2 and table 3)；
Ion pair, linear equation, the coefficient correlation of the determination of residual amount in the skim milk of table 2
Ion pair, linear equation, the coefficient correlation of the determination of residual amount in the whole milk of table 3
From table 2 and table 3, in the range of linearity investigated, the linearly dependent coefficient of each standard curve is higher,
Corresponding relations of the mass concentration x (μ g/kg) of energy each compound of accurate characterization and the ratio Y of peak area between.
(4) content of target non-protein nitrogen-containing compound is determined：
By prepare liquid under the conditions of default Liquid Chromatography-Tandem Mass Spectrometry sample introduction, according to testing result standard working curve
Corresponding non-protein nitrogen-containing compound in prepare liquid is quantified.
Corresponding non-protein nitrogen-containing compound should be in the range of linearity of instrument detection in prepare liquid.If one-time detection is completed
When the prepare liquid ionic liquid concentration that obtains beyond the investigation scope of primary standard curve, now should again prepare series concentration
Standard liquid, repaints standard curve, detects again.
In step (3) and (4), the mass spectrometry parameters of five kinds of non-protein nitrogen-containing compounds and internal standard compound are as shown in table 4.
The mass spectrometry parameters of 4 five kinds of non-protein nitrogen-containing compounds of table and internal standard compound
Note：Band " * " is quota ion pair.
In step (3) and (4), Liquid Chromatography-Tandem Mass Spectrometry condition is as shown in table 5：
The instrument condition parameter of the Liquid Chromatography-Tandem Mass Spectrometry of table 5
Under the conditions of above-mentioned Liquid Chromatography-Tandem Mass Spectrometry, judge to whether there is corresponding measured object, it is necessary to full in prepare liquid
The following condition of foot：1. the mass chromatography peak retention time for occurring in prepare liquid is consistent with standard hybrid working solution, it is allowed to deviation
Less than ± 2.5%；2. the relative ion abundance of the mass spectrometry ion pair (referring to table 4) of the compound corresponding to chromatographic peak with it is dense
The suitable standard hybrid working solution of degree is consistent, regulation of the relative ion abundance deviation no more than table 5；Meet two above condition
Then can determine that in prepare liquid containing corresponding non-protein nitrogen-containing compound.
To the ground former times Buddhist nun of 50 μ g/kg of each addition in the blank skim milk without above-mentioned five kinds of non-protein nitrogen-containing compounds
That, dicyandiamide, biuret, cyromazine and melamine, are processed by step (1) and step (2), obtain matrix solution, are obtained
Five kinds of MRM total ion current figures of non-protein nitrogen-containing compound, as shown in Figure 1.
It can be seen from figure 1 that the retention time phase listed in five kinds of chromatographic peak appearance times of non-protein nitrogen-containing compound and table 4
Unanimously, show that matrix solution does not influence the appearance time of each compound.
The quantitative limit of embodiment 2 is tested
According to Dicyclanil, dicyandiamide, biuret, the ammonia of ring third of 50 μ g/kg of each addition in milk (and whole milk)
Signal response intensity and signal to noise ratio when piperazine and melamine, are equal to 10 and calculate with S/N, addition Dicyclanil, dicyandiamide, contracting two
Five kinds of non-protein nitrogen-containing compounds in urea, cyromazine and melamine, the final measure for determining non-protein nitrogen-containing compound is determined
Amount limit, testing result is shown in Table 6.
Detection quantitative limit of the detection method of table 6 to each non-protein nitrogen-containing compound
From table 6, detection method of the invention is far below domestic and international minimum limitation to the detection quantitative limit of melamine
Standard (50 μ g/kg), while also possessing relatively low detection quantitative limit to other four kinds of non-protein nitrogen-containing compounds, shows the present invention
Detection method have stronger sensitivity and accuracy, can meet well trace detection requirement.
The recovery test of embodiment 3
To being added respectively in the blank skim milk (and whole milk) without above-mentioned five kinds of non-protein nitrogen-containing compounds
The Dicyclanil of known quantity, dicyandiamide, biuret, cyromazine and melamine, and it is de- to blank according to the method for embodiment 1
Fat milk (and whole milk) makees pre-treatment, according to the standard curve of embodiment 1, using the liquid chromatography tandem of embodiment 1
The content of each non-protein nitrogen-containing compound, carries out recovery test in Mass Spectrometry Conditions detection prepare liquid.
The rate of recovery (%)=(content in yield-sample)/mark-on amount × 100%；
Parallel repetition six times is tested every time, and recovery test result is as shown in table 7.
Five kinds of non-protein nitrogen-containing compound TIANZHU XINGNAO Capsuls and precision (n=6) in the sample of table 7
From table 7, five kinds of overall rate of recovery of non-protein nitrogen-containing compound are relative to mark between 78.5~101.8%
Quasi- deviation shows detection method of the invention for five kinds of nitrogenous chemical combination of non-protein in detection dairy products 1.3%~12.3%
Thing has good accuracy, reappearance, practicality.
Embodiment 4
Prepare the mixed of the Dicyclanil containing 12.5ng/mL, dicyandiamide, biuret, cyromazine and melamine respectively
Close solution, and method according to embodiment 1 makees pre-treatment to mixed solution, each non-protein nitrogen in prepare liquid after detection purification
The content of compound, calculates the rate of recovery.
Following comparative example is set simultaneously：
Comparative example 1：It is each non-in lower clear liquid when the ungrease treatment of detection n-hexane is completed using acetonitrile as protein precipitant
The content of albumen nitrogen-containing compound, calculates the rate of recovery；
Comparative example 2：Using acetonitrile as protein precipitant, purified using QuEChERS technologies, prepare liquid after detection purification
In each non-protein nitrogen-containing compound content, calculate the rate of recovery；
Comparative example 3：Using methyl alcohol as protein precipitant, purified using QuEChERS technologies, prepare liquid after detection purification
In each non-protein nitrogen-containing compound content, calculate the rate of recovery；
Comparative example 4：Using MCX SPE column purifications, each non-protein nitrogen-containing compound in prepare liquid after detection purification
Content, calculates the rate of recovery；
Comparative example 5：Using C18SPE column purification, each non-protein nitrogen-containing compound contains in prepare liquid after detection purification
Amount, calculates the rate of recovery；
Comparative example 6：Lower clear liquid is concentrated into after closely doing and is dissolved using acetonitrile, using HLB SPE column purifications, detection purification
Afterwards in prepare liquid each non-protein nitrogen-containing compound content, calculate the rate of recovery.
Result of calculation is shown in Table 8.
The rate of recovery of the embodiment 4 of table 8 and comparative example 1~6 compares
From table 8, the rate of recovery of comparative example 1 shows that n-hexane can not only effectively take off 93.1~101.8%
Fat, and have substantially no effect on the rate of recovery (i.e. target compound is not lost in).
The rate of recovery of comparative example 2 and comparative example 3 is basically identical, shows to make protein precipitant to returning using methyl alcohol or acetonitrile
Yield is substantially without influence；But the rate of recovery of comparative example 2 and comparative example 3 is below comparative example 6 and embodiment 4, shows QuEChERS
Purification techniques is poor to the rate of recovery of five kinds of non-protein nitrogen-containing compounds, and QuEChERS purification techniques is to dicyandiamide and ground former times Buddhist nun
Though you can reach 70%, 50% is less than to other three kinds of rate of recovery of compound.
Find out from the result of comparative example 4, MCX Solid phase extractions are to Dicyclanil, cyromazine, 3 changes of melamine
The rate of recovery 60-80% of compound, other two compound rate of recovery are less desirable, and only 30% or so.
Though the rate of recovery of comparative example 5 is better than comparative example 4, and the rate of recovery of five kinds of compounds is higher, and it is right to be still below
Ratio 6.
Embodiment 4 substitutes the acetonitrile in comparative example 6 using ammoniacal liquor acetonitrile, further increases the rate of recovery.
Embodiment 5
It is 1 in volume ratio:9 0.15% aqueous formic acid (ammonium acetate containing 5mM)-acetonitrile mixture respectively adds ground former times Buddhist nun
That, dicyandiamide, biuret, cyromazine and melamine to final concentration of 12.5ng/mL, using acetonitrile and 0.15% formic acid water
(mobile phase initial soln is with constant volume solution with [i.e. 0.15% aqueous formic acid used as mobile phase for solution (ammonium acetate containing 5mM)
The volume ratio of (ammonium acetate containing 5mM)-acetonitrile is 1:9], Liquid Chromatography-Tandem Mass Spectrometry is made using the method for the step of embodiment 1 (4),
Situations such as investigating separation appearance, spectral peak peak shape and the signal intensity of each compound (see Fig. 2).
Following comparative's example is set simultaneously：
Comparative example 7：It is 1 in volume ratio:Dicyclanil, double cyanogen are added in 9 0.15% aqueous formic acid-acetonitrile mixture
Amine, biuret, cyromazine and melamine use acetonitrile and 0.15% aqueous formic acid to final concentration of 12.5ng/mL
Used as mobile phase, mobile phase initial soln is with constant volume solution with (see Fig. 3)；
Comparative example 8：It is 1 in volume ratio:Dicyclanil, double cyanogen are added in 9 0.15% aqueous formic acid-acetonitrile mixture
Amine, biuret, cyromazine and melamine (are contained to final concentration of 12.5ng/mL using acetonitrile and 0.15% aqueous formic acid
5mM ammonium acetates) used as mobile phase, (mobile phase initial soln is that volume ratio is 1:9 0.15% aqueous formic acid (acetic acid containing 5mM
Ammonium)-acetonitrile mixture) (see Fig. 4).
From Fig. 2 and Fig. 3, compared with comparative example 7,5mM acetic acid is added in the constant volume solution and mobile phase of embodiment 5
Ammonium can significantly improve the signal intensity of each compound, and wherein Dicyclanil intensity improves nearly 50 times.
From Fig. 2 and Fig. 4, comparative example 8 is inconsistent with mobile phase initial soln due to constant volume solution, causes Dicyclanil
There is " bifurcated " peak in spectral peak peak shape；And embodiment 5 is consistent with mobile phase initial soln due to constant volume solution, spectral peak peak shape is preferable.
In 0.15% aqueous formic acid, when the addition concentration of ammonium acetate is in 3~10mM, the separation appearance of each compound,
Situations such as spectral peak peak shape and signal intensity, is essentially identical.
Claims (10)
1. the method that liquid chromatography tandem mass spectrometry detects various non-protein nitrogen-containing compound contents in milk simultaneously, its feature exists
In comprising the following steps：
(1) to protein precipitant is added in testing sample, it is vortexed, centrifugation takes supernatant；
(2) to grease removal agent is added in the supernatant, it is vortexed, centrifugation, reject fat oxidant layer uses ammoniacal liquor acetonitrile after lower clear liquid is concentrated
Dissolving, vortex, obtain liquid to be clean；
(3) liquid to be clean is crossed into HLB solid-phase extraction columns, it is 1 to collect eluent and volume ratio is used after being concentrated:9
0.15% aqueous formic acid-acetonitrile mixture dissolving, constant volume, filtering with microporous membrane obtain prepare liquid；
Contain 3~10mM ammonium acetates in 0.15% aqueous formic acid；
(4) prepare nitrogenous containing at least four non-protein in Dicyclanil, dicyandiamide, biuret, cyromazine and melamine
The standard mixed solution of compound, gradient dilution is carried out by standard mixed solution, obtains standard hybrid working solution series；
(5) the serial and described prepare liquid of the standard hybrid working solution is divided under the conditions of default Liquid Chromatography-Tandem Mass Spectrometry
Other sample introduction, the testing result according to standard hybrid working solution series makes standard curve, further according to standard curve and prepare liquid
Testing result calculate the content of corresponding non-protein nitrogen-containing compound in prepare liquid；
Wherein, liquid phase chromatogram condition is：
Chromatographic column：Waters XBridge HILIC, 4.6mm × 150mm, 3.5 μm；
Mobile phase A：0.15% aqueous formic acid, containing 3~10mM ammonium acetates；
Mobile phase B：Acetonitrile；
Flow velocity：0.4mL/min；
Sample size：10μL；
Column temperature：40℃；
Gradient elution program：①0min：10%A, 90%B；
②3.0min：50%A, 50%B；
③8.0min：50%A, 50%B；
④9.0min：10%A, 90%B；
⑤20.0min：10%A, 90%B；
Mass Spectrometry Conditions are：
Ion gun：Electric spray ion source；
Scan mode：Cation is scanned；
Monitoring mode：Multiple-reaction monitoring；
Electron spray voltage：3500V；
Ion source temperature：250℃；
Dry temperature degree：High pure nitrogen, 250 DEG C；
Dry gas stream amount：High pure nitrogen, 16L/min；
Atomization gas pressure：High pure nitrogen, 275.8kPa；
Sheath temperature degree：High pure nitrogen, 350 DEG C；
Sheath throughput：High pure nitrogen, 10L/min；
Mass spectrometry parameters are：
6. Dicyclanil：Quota ion pair is 191.1/150.0, and collision energy is 21V；Qualitative ion pair is 191.1/109.1,
Collision energy is 31V；
7. dicyandiamide：Quota ion pair is 85.1/68.1, and collision energy is 20V；Qualitative ion pair is 85.1/43.1, impact energy
It is 22V to measure；
8. biuret：Quota ion pair is 104.2/61.0, and collision energy is 9V；Qualitative ion pair is 104.2/44.1, collision
Energy is 37V；
9. cyromazine：Quota ion pair is 167.2/85.1, and collision energy is 20V；Qualitative ion pair is 167.2/125.1,
Collision energy is 18V；
10. melamine：Quota ion pair is 127.1/85.1, and collision energy is 19V；Qualitative ion pair is 127.1/68.1, is touched
Energy is hit for 36V.
2. the method for claim 1, it is characterised in that in step (1), first to adding melamine in the testing sample
Amine-15N3Internal standard compound, then add protein precipitant.
3. method as claimed in claim 2, it is characterised in that the melamine-15N3The addition of internal standard compound be 50~
200ng。
4. method as claimed in claim 2 or claim 3, it is characterised in that the melamine-15N3The mass spectrometry parameters of internal standard compound are：
Ion pair is 130.2/87.1, and collision energy is 19V.
5. method as claimed in claim 1 or 2, it is characterised in that in step (1), the protein precipitant is acetonitrile.
6. method as claimed in claim 5, it is characterised in that acetonitrile is (15~20) with the mixed volume ratio of testing sample：
1。
7. method as claimed in claim 1 or 2, it is characterised in that in step (2), the grease removal agent is n-hexane.
8. method as claimed in claim 7, it is characterised in that the mixed volume ratio of n-hexane and the supernatant for (1~
1.5)：2.
9. method as claimed in claim 1 or 2, it is characterised in that in step (2), after the lower clear liquid is concentrated with 3~
10% ammoniacal liquor acetonitrile dissolves.
10. method as claimed in claim 1 or 2, it is characterised in that in step (3), second in 0.15% aqueous formic acid
The concentration of sour ammonium is 5mM.
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| CN107621399B (en) * | 2016-07-14 | 2021-04-27 | 北京三元食品股份有限公司 | Method for detecting oligosaccharide in breast milk |
| CN112505165A (en) * | 2020-07-10 | 2021-03-16 | 中检科（北京）测试技术有限公司 | Method for measuring residual quantity of dicyandiamide in dairy product |
| CN113252818B (en) * | 2021-07-07 | 2021-10-26 | 裕菁科技(上海)有限公司 | Method for quantifying and evaluating compounds of same series by adopting reference sample |
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| US3782075A (en) * | 1972-04-07 | 1974-01-01 | Du Pont | Completely porous microspheres for chromatographic uses |
| DE3738675C1 (en) * | 1987-11-13 | 1988-12-08 | Sueddeutsche Kalkstickstoff | Method for the determination of dicyandiamide in plants or parts of plants |
| NO176136C (en) * | 1988-05-18 | 1995-02-08 | Nippon Carbide Kogyo Kk | Process for the preparation of cyanamide |
| CN101387609B (en) * | 2008-10-20 | 2010-09-15 | 中国农业大学 | Method for rapidly detecting melamine |
| CN101576543A (en) * | 2009-04-21 | 2009-11-11 | 新疆出入境检验检疫局检验检疫技术中心 | Method for fast detecting melamine in dry milk |
| CN102998305A (en) * | 2011-09-19 | 2013-03-27 | 青岛大学 | Method for detecting melamine in raw milk by utilizing chromogenic reaction |
| CN102435699B (en) * | 2011-09-22 | 2014-12-24 | 明一（福建）婴幼儿营养品有限公司 | Method for rapidly determining melamine in milk and dairy products by liquid chromatography-tandem mass spectrometry |
| CN105021737B (en) * | 2015-08-10 | 2017-04-05 | 云南省食品药品检验所 | It is a kind of at the same detect milk product in dicyandiamide and content of melamine method |
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