CN101576543A - Method for fast detecting melamine in dry milk - Google Patents
Method for fast detecting melamine in dry milk Download PDFInfo
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- CN101576543A CN101576543A CNA2009101132948A CN200910113294A CN101576543A CN 101576543 A CN101576543 A CN 101576543A CN A2009101132948 A CNA2009101132948 A CN A2009101132948A CN 200910113294 A CN200910113294 A CN 200910113294A CN 101576543 A CN101576543 A CN 101576543A
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Abstract
The invention discloses a method for fast detecting melamine in dry milk. Melamine in dry milk is extracted under ultrasonic by acetic acid-acetonitrile-aqueous solution; after centrifugation and filtration, filter liquor is purified by a mixed type cation exchange solidoid extraction column and is blow-dried by nitrogen, microwave heating derivatization reaction is carried out, the reaction time is shortened, and by optimizing the ion selection of an ion selection monitoring mode, the influence of substrate is eliminated, the capacity of resisting disturbance is enhanced, the detection limit is lowered, and the quantitative reproducibility is improved. Thus, the simple, fast, sensitive, accurate, low-cost and durable verification and quantification method for detecting melamine in dry milk is established, and can be widely applied to the technical field of detecting melamine in food represented by dry milk.
Description
Invention field
The invention belongs to the food safety detection field, be specifically related to the detection method of hazard residue thing in the food, particularly relate to the method field of melamine in a kind of fast detecting milk powder.
Background technology
Melamine (Melamine, MEL), formal name used at school 1,3,5-triamido-2,4, the 6-triazine (1,3,5-Triamine-2,4,6-Triazine), molecular formula: C
3N
3(NH
2)
3, molecular weight 126.12.Be called for short triamine, claim 2 again, 4,6-triamido urea, melamine, melamine, cyanogen urea triamide etc., white crystalline powder, tasteless, be a kind of triazines nitrogen-containing heterocycle compound, important Organic Chemicals is widely used in the production of plastics, coating, bonding agent, packaging material for food, sanitizer, chemical fertilizer and pesticide.The topmost purposes of melamine is as the raw material of producing melamine formaldehyde resin (MF).Because the defective of food and feed industry protein content method of testing, melamine also often are used as food additives by illegal businessman, with the protein content index in the lifting food inspection, so melamine also is " extract of protein " by person.Data shows that melamine may enter into food from approach such as environment, packaging materials for food, and its content is very low.Melamine is not a raw-food material, neither food additives, forbid that the people is for adding in the food.
Protein mainly is made up of amino acid, and its nitrogen content generally is no more than 30%, and the molecular formula nitrogen content of melamine is about 66.7%.General protein method of testing " Kjeldahl " is to estimate protein content by measuring nitrogen content, therefore, adds melamine and can make that the protein test content of food is higher, thereby make the test of poor quality food by Food Inspection mechanism.But the edible food that has added melamine can cause poisoning and kidney stone.2007, the U.S. reported that pet cat, dog contain the dead incident of melamine feed because of eating, and post-survey shows: the gluten powder of 6.6% melamine that mixed is that pet food causes the reason of poisoning.FDA (Food and Drug Adminstration) (FDA) has worked out the corresponding method of inspection subsequently.On September 11st, 2008, relevant department discloses, and contains melamine in some milk powder, has caused some infants to contain melamine milk powder and the kidney stone symptom occurs because of edible, and all rescue measures proved ineffectual and death for minority infant.At some liquid milk, dairy products and contain and also detect melamine in the dairy products, bring huge impact for whole food service industry subsequently, social influence is extremely bad.In detecting the product of melamine, Shijiazhuang Sanlu board baby formula milk powder content of melamine is very high, the wherein the highest 2563mg/kg that reaches.In October, 2008, national departments concerned has been formulated " melamine detection method in raw milk and the dairy products " national standard (GB/T223888-2008) with reference to FDA (Food and Drug Adminstration) (FDA) and U.S. food chemicals code (FCC) melamine detection method.On October 7th, 2008,5 departments such as the Ministry of Public Health have announced dairy products and have contained melamine temporary control and education value of limiting the quantity of in the dairy products that the bulletin regulation: one, the value of limiting the quantity of of melamine is 1mg/kg in the baby formula milk powder, and the product that is higher than 1mg/kg must not be sold without exception.Two, the value of limiting the quantity of of melamine is 2.5mg/kg in liquid milk (comprising raw milk), milk powder, other prescription emulsifiable powders, and the product that is higher than 2.5mg/kg must not be sold without exception.Three, the value of limiting the quantity of that contains melamine in other food 15% or more of breast is 2.5mg/kg, and the product that is higher than 2.5mg/kg must not be sold without exception.
The method of measuring melamine has high performance liquid chromatography (HPLC), gas chromatography-mass spectrography (GC-MS) and high performance liquid chromatography-tandem mass (HPLC-MS/MS).The sensitivity of HPLC is lower and structural information can't be provided, general Chang Zuowei screening technique; GC-MS, HPLC-MS/MS can carry out confirmatory analysis.GB/T22388-2008 " melamine detection method in raw milk and the dairy products " has stipulated three kinds of assay methods of melamine in raw milk and the dairy products, i.e. HPLC method, GC-MS method, HPLC-MS/MS method.
Among the GB/T22388-2008 " melamine detection method in raw milk and the dairy products " gas chromatography-mass spectrography (GC-MS) method sample pre-treatments loaded down with trivial details, need to consume a large amount of reagent, time-consuming, cost is high, and each test sample is limited, the examination of incompatible a large amount of samples, the scene that also is not suitable for is detected fast, can not high-throughout test sample in melamine.Therefore; explore and study easy, quick, sensitive, accurately, the method for melamine in low-cost, the durable detection food, for safeguarding food security; protection people health and consumer's legitimate rights and interests promote that the fast development of food service industry is all significant.
Summary of the invention
Loaded down with trivial details at gas chromatography-mass spectrography (GC-MS) method sample pre-treatments among the GB/T22388-2008 " melamine detection method in raw milk and the dairy products ", need to consume a large amount of reagent, time-consuming, cost is high, and each test sample is limited, the examination of incompatible a large amount of samples, the scene that also is not suitable for is detected fast, can not high-throughout detection milk powder in the defective of melamine.
The object of the present invention is to provide a kind of the easy, quick of melamine in the milk powder of measuring, sensitive, accurately, low-cost, durable conclusive evidence and quantivative approach.
The present invention specifically provides a kind of conclusive evidence and quantivative approach of measuring melamine in the milk powder.
Technical scheme of the present invention: by having set up the method for melamine in the GC-MS method mensuration milk powder, with the melamine in acetate-acetonitrile-aqueous solution ultrasonic Extraction milk powder, filter centrifugal back, and filtrate is through mixed type cation exchange Solid-Phase Extraction column purification, and nitrogen dries up, the microwave heating derivative reaction, shorten the reaction time,, eliminate the matrix influence by to selecting the optimization of ion monitoring model selection ion, strengthen antijamming capability, reduce detectability and improve quantitative reappearance.Thereby set up a kind of conclusive evidence and quantivative approach that melamine is easy, quick, sensitive, accurate, low-cost, durable in the cream biscuit of measuring.
The present invention specifically provides a kind of method step of melamine of measuring in the milk powder as follows:
Take by weighing 0.50 gram milk powder sample in 15mL tool vice polypropylene graduated centrifuge tube, add 10mL acetate-acetonitrile-aqueous solution jolting, acetate-acetonitrile-aqueous solution is according to volume ratio preparation in 1: 70: 29, ultrasonic Extraction 10-30min, the centrifugal 5-10min of 4500r/min, filter after solid-phase extraction column activates with the methyl alcohol of 3-5mL successively, the 3-5mL water balance, accurately pipette 4-6mL filtrate upper prop, use the 0.1-0.3mol/L hydrochloric acid solution of 3-5mL successively, the methyl alcohol drip washing of 3-5mL dries up pillar with rubber pipette bulb, with 3-5mL5% ammoniacal liquor methanol-eluted fractions, collect eluent with 5mL scale test tube, 45-75 ℃ of nitrogen blow be concentrated into do after, add 200 μ L pyridines and 200 μ L derivatization reagents, seal with the glass ground joint plug, under the high-grade condition of 800W microwave, react 2-8min, cooling is transferred in the 1.5mL high-recovery sample bottle, and air feed phase chromatography-mass spectroscopy is measured.
Known in the art, melamine is slightly soluble in water (3.1g/L, 20 ℃), ethanol, weak acid and weak base etc., dissolves in methyl alcohol, pyridine, dimethyl sulfoxide (DMSO), and aqueous solution is alkalescent.Melamine belongs to polar compound, alkali compounds, therefore extracts stronger organic solvent or the buffer solution of mixed solution, polarity that solvent is generally all selected dilute acid soln, organic solvent and water for use.The present invention selects acetate-acetonitrile-aqueous solution for extracting solvent, add acetate and help improving the melamine percentage extraction, add acetonitrile and help precipitating water-solubility protein, reduce and disturb, acetate-acetonitrile-aqueous solution that the present invention adopts can effectively precipitate protein and the part fat in the milk sample, filter rapidly the filtrate clarification.
The present invention passes through selecting the ion-selective optimization of ion monitoring pattern (SIM), effectively eliminating complicated substrate disturbs: the characteristic ion of melamine is 99,171,197,285,327,342, and the characteristic ion that the GC-MS method is used among the GB/T22388-2008 " melamine detection method in raw milk and the dairy products " is 99,171,327,342.Analysis through to milk powder mark-on sample contains 99,171 fragment ions in the sample substrate, quantitative measurement is disturbed greatly, and the present invention finally characteristic ion of selected melamine is 197,285,327,342, adopts the incompatible quantitative and conclusive evidence of above-mentioned ion set.
Well known, derivative reaction is generally 70 ℃ of heating 45min down, and the present invention determines to adopt under the high-grade 800W condition of microwave through repetition test, and derivatization 5min has shortened the reaction time greatly.
By implementing the concrete technical indicator of the present invention, realize content of the present invention, can reach following beneficial effect.
1. high-recovery: adopt acetate-acetonitrile-aqueous solution to extract melamine in the sample among the present invention, make melamine be attracted on the cation exchange column with the positive ion attitude, come drip washing with 0.1mol/L salt acid substitution water, both can make melamine keep the positive ion attitude to be attracted on the cation exchange column, can effectively remove the oil-soluble impurities of water-solubility impurity, inorganic salts and nonionicization in the sample again, can reduce injection port and ionogenic pollution, the melamine recovery is between 93-107%.
2. degree of accuracy height: relative standard deviation is less than 9.2%.
3. time for sample pretreatment is short: 2 people can handle 30 duplicate samples in 180min, can realize melamine in the sample is carried out high throughput testing.
4. precipitating proteins is effective: the present invention is preferred through innovation, finally selects acetate-acetonitrile-aqueous solution for extracting solvent, and effectively protein in the deposit sample and part fat filter rapidly, the filtrate clarification.
5. good purification: sample mark-on spectrogram " totally ", noiseless at the melamine derivative appearance time, be beneficial to accurate conclusive evidence and quantitatively.
6. the sample sample weighting amount is few, the solvent use amount is few, pollute little, environmental protection: the sample sample weighting amount only is 1 gram, by to extracting the screening and the optimization of solvent, select the combination of extraction efficiency best solvent, every part of cream biscuit sample solvent use amount only is 22mL, and do not use the chloride solvent, discarded object is few.
7. flow process is simple: strong operability, be easy to grasp, and need not well trained and just can finely finish than high professional qualification.
8. use glassware seldom: only need to use 15mL tool vice polypropylene graduated centrifuge tube, 5mL scale test tube, 1.5mL high-recovery sample bottle.
9. institute's operative installations is simple in the specimen preparation process: only need miniature ultrasonic ripple washer, compact centrifuge, 12 hole solid-phase extraction devices, Nitrogen evaporator, turbine mixer etc., and easy to utilize.
10. requisite space is little, just can finish in the small test chamber: the pre-treatment of sample cream biscuit can be finished in 10 squares room, is easy to realize field test.
11. cost is low: the reagent that uses is the laboratory common agents, and low price is easy to buying, and consumption is few.
12. the present invention passes through selecting the ion-selective optimization of ion monitoring (SIM) pattern, effectively eliminating complicated substrate disturbs: the characteristic ion of selected melamine is 197,285,327,342, adopt the incompatible quantitative and conclusive evidence of above-mentioned ion set, for this invention is initiative.
13. the present invention adopts the microwave heating derivative reaction first, shorten the reaction time: derivative reaction is generally 70 ℃ of heating 45min down, and the present invention determines to adopt under the high-grade 800W condition of microwave through overtesting, derivatization 5min, shortened the reaction time greatly, for this invention is initiative.
Description of drawings:
Figure 1 shows that the melamine derivative mass spectrogram;
Figure 2 shows that and add 1.0mg/kg melamine milk powder sample SIM mass spectrogram.
Embodiment
Below, for embodiment the present invention is described, still, the present invention is not limited to following embodiment.In addition, in following explanation, if no special instructions, % all refers to percentage by weight.
Equipment and material have among the present invention:
Agilent7890A/5975C gas chromatograph-mass spectrometer (GCMS) (U.S. Agilent company) is equipped with 7683 automatic samplers, enhanced chemical workstation; 18790 xeothermic sample concentration devices (U.S. Pierce company); Micro-wave oven (Glanz company, 800W); Compact centrifuge; Ultrasonic cleaner; 1.5mL screw thread mouth high-recovery sample bottle (U.S. Agilent company, P/N5183-2030).
The melamine standard items: purity is 99.5%, and Chemservice company provides;
Mixed type cation exchange solid-phase extraction column, 60mg/3mL, Beijing Xiang company of shaking; Derivatization reagent: N, the two trimethyls of O--silica-based trifluoroacetamide (BSTF4)+1% trimethyl chlorosilane (TMCS), German Macherey Nagel company; Pyridine (top grade is pure); Methyl alcohol, acetonitrile (chromatographically pure); Glacial acetic acid (analyzing pure); Hydrochloric acid (analyzing pure); Ammoniacal liquor (analyzing pure); Water is ultrapure water.
Embodiment one:
Take by weighing 0.50 gram milk powder sample in 15mL tool vice polypropylene graduated centrifuge tube, add 10mL acetate-acetonitrile-aqueous solution jolting, acetate-acetonitrile-aqueous solution is according to volume ratio preparation in 1: 70: 29, ultrasonic Extraction 10min, the centrifugal 5min of 4500r/min, filter after solid-phase extraction column activates with the methyl alcohol of 3mL successively, the 3mL water balance accurately pipettes 4mL filtrate upper prop, use the 0.1mol/L hydrochloric acid solution of 3mL successively, the methyl alcohol drip washing of 3mL dries up pillar with rubber pipette bulb, with 3mL5% ammoniacal liquor methanol-eluted fractions, collect eluent with 5mL scale test tube, 45 ℃ of nitrogen blow be concentrated into do after, add 200 μ L pyridines and 200 μ L derivatization reagents, seal with the glass ground joint plug, under the high-grade condition of 800W microwave, react 2min, cooling is transferred in the 1.5mL high-recovery sample bottle, and air feed phase chromatography-mass spectroscopy is measured.
Adopt the melamine in acetate-acetonitrile-aqueous solution extraction sample, make melamine be attracted on the cation exchange column with the positive ion attitude, come drip washing with 0.1mol/L salt acid substitution water, both can make melamine keep the positive ion attitude to be attracted on the cation exchange column, can effectively remove the oil-soluble impurities of water-solubility impurity, inorganic salts and nonionicization in the sample again, can reduce injection port and ionogenic pollution, obtain the high recovery, the melamine recovery reaches 93%, relative standard deviation is less than 9.2%, the degree of accuracy height; This experiment realizes that 2 people can handle 30 duplicate samples in 180min, has realized melamine in the sample is carried out high throughput testing, and time for sample pretreatment is short; Select acetate-acetonitrile-aqueous solution for extracting solvent, filter rapidly, the filtrate clarification, precipitating proteins is effective; Sample mark-on spectrogram " totally ", noiseless at the melamine derivative appearance time, be beneficial to accurate conclusive evidence and quantitative, good purification; Do not use the chloride solvent, every duplicate samples solvent use amount only is 22mL, and the sample sample weighting amount is few, and the solvent use amount is few, and discarded object is few, pollute little, environmental protection; Use glassware seldom, only need to use 15mL tool vice polypropylene graduated centrifuge tube, 5mL scale test tube, 1.5mL high-recovery sample bottle; Institute's operative installations is simple in the specimen preparation process, only needs miniature ultrasonic ripple washer, compact centrifuge, 12 hole solid-phase extraction devices, Nitrogen evaporator, turbine mixer etc., and is easy to utilize; Requisite space is little, just can finish in the small test chamber: the pre-treatment of sample can be finished in 10 squares room, is easy to realize field test; The reagent that uses is the laboratory common agents, and low price is easy to buying, and consumption is few, and cost is low.
The present invention passes through selecting the ion-selective optimization of ion monitoring (SIM) pattern, effectively eliminating complicated substrate disturbs: the characteristic ion of melamine is 99,171,197,285,327,342, and the characteristic ion that the GC-MS method is used among the GB/T22388-2008 " melamine detection method in raw milk and the dairy products " is 99,171,327,342.Analysis through to milk powder mark-on sample contains 99,171 fragment ions in the sample substrate, big to the quantitative measurement interference, the characteristic ion of this final selected melamine is 197,285,327,342, adopts the incompatible quantitative and conclusive evidence of above-mentioned ion set; The present invention adopts the microwave heating derivative reaction, has shortened the reaction time greatly, more than 2 for this invention for the initiative.
Embodiment two:
Take by weighing 0.50 gram milk powder sample in 15mL tool vice polypropylene graduated centrifuge tube, add 10mL acetate-acetonitrile-aqueous solution jolting, acetate-acetonitrile-aqueous solution is according to volume ratio preparation in 1: 70: 29, ultrasonic Extraction 30min, the centrifugal 10min of 4500r/min, filter after solid-phase extraction column activates with the methyl alcohol of 5mL successively, the 5mL water balance accurately pipettes 6mL filtrate upper prop, use the 0.3mol/L hydrochloric acid solution of 5mL successively, the methyl alcohol drip washing of 5mL dries up pillar with rubber pipette bulb, with 5mL5% ammoniacal liquor methanol-eluted fractions, collect eluent with 5mL scale test tube, 75 ℃ of nitrogen blow be concentrated into do after, add 200 μ L pyridines and 200 μ L derivatization reagents, seal with the glass ground joint plug, under the high-grade condition of 800W microwave, react 8min, cooling is transferred in the 1.5mL high-recovery sample bottle, and air feed phase chromatography-mass spectroscopy is measured.
Adopt the melamine in acetate-acetonitrile-aqueous solution extraction sample, both can make melamine keep the positive ion attitude to be attracted on the cation exchange column, can effectively remove the oil-soluble impurities of water-solubility impurity, inorganic salts and nonionicization in the sample again, can reduce injection port and ionogenic pollution, obtain the high recovery, the melamine recovery reaches 107%, relative standard deviation is less than 9.2%, the degree of accuracy height, and filter rapidly, the filtrate clarification, precipitating proteins is effective.
This tests 2 people can handle 30 duplicate samples in 180min, realized melamine in the sample is carried out high throughput testing, and time for sample pretreatment is short; Sample mark-on spectrogram " totally ", noiseless at the melamine derivative appearance time, be beneficial to accurate conclusive evidence and quantitative, good purification; Do not use the chloride solvent, every duplicate samples solvent use amount only is 22mL, and the sample sample weighting amount is few, and the solvent use amount is few, and discarded object is few, pollute little, environmental protection; Use glassware seldom, only need to use 15mL tool vice polypropylene graduated centrifuge tube, 5mL scale test tube, 1.5mL high-recovery sample bottle; Institute's operative installations is simple in the specimen preparation process, only needs miniature ultrasonic ripple washer, compact centrifuge, 12 hole solid-phase extraction devices, Nitrogen evaporator, turbine mixer etc., and is easy to utilize; Requisite space is little, just can finish in the small test chamber: the pre-treatment of sample can be finished in 10 squares room, is easy to realize field test; The reagent that uses is the laboratory common agents, and low price is easy to buying, and consumption is few, and cost is low.
The present invention passes through selecting the ion-selective optimization of ion monitoring (SIM) pattern, effectively eliminating complicated substrate disturbs, through analysis to milk powder mark-on sample, contain 99,171 fragment ions in the sample substrate, big to the quantitative measurement interference, the characteristic ion of selected melamine is that 197,285,327,342 combinations come quantitative and conclusive evidence.
Embodiment three:
Take by weighing 0.50 gram milk powder sample in 15mL tool vice polypropylene graduated centrifuge tube, add 10mL acetate-acetonitrile-aqueous solution jolting, acetate-acetonitrile-aqueous solution is according to volume ratio preparation in 1: 70: 29, ultrasonic Extraction 20min, the centrifugal 8min of 4500r/min, filter after solid-phase extraction column activates with the methyl alcohol of 4mL successively, the 4mL water balance accurately pipettes 5mL filtrate upper prop, use the 0.2mol/L hydrochloric acid solution of 4mL successively, the methyl alcohol drip washing of 4mL dries up pillar with rubber pipette bulb, with 4mL5% ammoniacal liquor methanol-eluted fractions, collect eluent with 5mL scale test tube, 60 ℃ of nitrogen blow be concentrated into do after, add 200 μ L pyridines and 200 μ L derivatization reagents, seal with the glass ground joint plug, under the high-grade condition of 800W microwave, react 4min, cooling is transferred in the 1.5mL high-recovery sample bottle, and air feed phase chromatography-mass spectroscopy is measured.
This experiment is adopted acetate-acetonitrile-aqueous solution to extract melamine in the sample and is obtained the high recovery and reach 96%, and relative standard deviation is less than 9.2%, the degree of accuracy height, and filter rapidly, the filtrate clarification, precipitating proteins is effective; Realized melamine in the sample is carried out high throughput testing, time for sample pretreatment is short; Sample mark-on spectrogram " totally ", noiseless at the melamine derivative appearance time, be beneficial to accurate conclusive evidence and quantitative, good purification; Do not use the chloride solvent, the solvent use amount is few, pollutes little; Institute's operative installations is simple in the specimen preparation process, and requisite space is little, just can finish in the small test chamber, easy to utilize and realization field test; The reagent that uses is the laboratory common agents, and low price is easy to buying, and consumption is few, and cost is low; The present invention eliminates the matrix influence by to selecting the optimization of ion monitoring (SIM) model selection ion, strengthens antijamming capability, reduces detectability and improves quantitative reappearance.
Embodiment four:
Take by weighing 0.50 gram milk powder sample in 15mL tool vice polypropylene graduated centrifuge tube, add 10mL acetate-acetonitrile-aqueous solution jolting, acetate-acetonitrile-aqueous solution is according to volume ratio preparation in 1: 70: 29, ultrasonic Extraction 25min, the centrifugal 9min of 4500r/min, filter after solid-phase extraction column activates with the methyl alcohol of 4mL successively, the 3.5mL water balance accurately pipettes 4.5mL filtrate upper prop, use the 0.2mol/L hydrochloric acid solution of 3.5mL successively, 3.5mL methyl alcohol drip washing, with rubber pipette bulb pillar is dried up, with 3mL5% ammoniacal liquor methanol-eluted fractions, collect eluent with 5mL scale test tube, 46 ℃ of nitrogen blow be concentrated into do after, add 200 μ L pyridines and 200 μ L derivatization reagents, seal with the glass ground joint plug, under the high-grade condition of 800W microwave, react 3min, cooling is transferred in the 1.5mL high-recovery sample bottle, and air feed phase chromatography-mass spectroscopy is measured.
This experiment is adopted acetate-acetonitrile-aqueous solution to extract melamine in the sample and is obtained the high recovery and reach 98%, and relative standard deviation is less than 9.2%, the degree of accuracy height, and filter rapidly, the filtrate clarification, precipitating proteins is effective; Realized melamine in the sample is carried out high throughput testing, time for sample pretreatment is short; Sample mark-on spectrogram " totally ", noiseless at the melamine derivative appearance time, be beneficial to accurate conclusive evidence and quantitative, good purification; Do not use the chloride solvent, the solvent use amount is few, pollutes little; Institute's operative installations is simple in the specimen preparation process, and requisite space is little, just can finish in the small test chamber, easy to utilize and realization field test; The reagent that uses is the laboratory common agents, and low price is easy to buying, and consumption is few, and cost is low; The present invention eliminates the matrix influence by to selecting the optimization of ion monitoring (SIM) model selection ion, strengthens antijamming capability, reduces detectability and improves quantitative reappearance.
Embodiment five:
Take by weighing 0.50 gram milk powder sample in 15mL tool vice polypropylene graduated centrifuge tube, add 10mL acetate-acetonitrile-aqueous solution jolting, acetate-acetonitrile-aqueous solution is according to volume ratio preparation in 1: 70: 29, ultrasonic Extraction 29min, the centrifugal 9min of 4500r/min, filter after solid-phase extraction column activates with the methyl alcohol of 5mL successively, the 5mL water balance accurately pipettes 5.5mL filtrate upper prop, use the 0.3mol/L hydrochloric acid solution of 4.5mL successively, the methyl alcohol drip washing of 5mL dries up pillar with rubber pipette bulb, with 5mL5% ammoniacal liquor methanol-eluted fractions, collect eluent with 5mL scale test tube, 74 ℃ of nitrogen blow be concentrated into do after, add 200 μ L pyridines and 200 μ L derivatization reagents, seal with the glass ground joint plug, under the high-grade condition of 800W microwave, react 7min, cooling is transferred in the 1.5mL high-recovery sample bottle, and air feed phase chromatography-mass spectroscopy is measured.
This experiment is adopted acetate-acetonitrile-aqueous solution to extract melamine in the sample and is obtained the high recovery and reach 105%, and relative standard deviation is less than 9.2%, the degree of accuracy height, and filter rapidly, the filtrate clarification, precipitating proteins is effective; Realized melamine in the sample is carried out high throughput testing, time for sample pretreatment is short; Sample mark-on spectrogram " totally ", noiseless at the melamine derivative appearance time, be beneficial to accurate conclusive evidence and quantitative, good purification; Do not use the chloride solvent, the solvent use amount is few, pollutes little; Institute's operative installations is simple in the specimen preparation process, and requisite space is little, just can finish in the small test chamber, easy to utilize and realization field test; The reagent that uses is the laboratory common agents, and low price is easy to buying, and consumption is few, and cost is low; The present invention eliminates the matrix influence by to selecting the optimization of ion monitoring (SIM) model selection ion, strengthens antijamming capability, reduces detectability and improves quantitative reappearance.
Embodiment six:
Take by weighing 0.50 gram milk powder sample in 15mL tool vice polypropylene graduated centrifuge tube, add 10mL acetate-acetonitrile-aqueous solution jolting, acetate-acetonitrile-aqueous solution is according to volume ratio preparation in 1: 70: 29, ultrasonic Extraction 11min, the centrifugal 7min of 4500r/min, filter after solid-phase extraction column activates with the methyl alcohol of 3mL successively, the 3mL water balance accurately pipettes 4.5mL filtrate upper prop, use the 0.1mol/L hydrochloric acid solution of 3mL successively, the methyl alcohol drip washing of 3mL dries up pillar with rubber pipette bulb, with 3mL5% ammoniacal liquor methanol-eluted fractions, collect eluent with 5mL scale test tube, 50 ℃ of nitrogen blow be concentrated into do after, add 200 μ L pyridines and 200 μ L derivatization reagents, seal with the glass ground joint plug, under the high-grade condition of 800W microwave, react 6min, cooling is transferred in the 1.5mL high-recovery sample bottle, and air feed phase chromatography-mass spectroscopy is measured.
This experiment is adopted acetate-acetonitrile-aqueous solution to extract melamine in the sample and is obtained the high recovery and reach 101%, and relative standard deviation is less than 9.2%, the degree of accuracy height, and filter rapidly, the filtrate clarification, precipitating proteins is effective; Realized melamine in the sample is carried out high throughput testing, time for sample pretreatment is short; Sample mark-on spectrogram " totally ", noiseless at the melamine derivative appearance time, be beneficial to accurate conclusive evidence and quantitative, good purification; Do not use the chloride solvent, the solvent use amount is few, pollutes little; Institute's operative installations is simple in the specimen preparation process, and requisite space is little, just can finish in the small test chamber, easy to utilize and realization field test; The reagent that uses is the laboratory common agents, and low price is easy to buying, and consumption is few, and cost is low; The present invention eliminates the matrix influence by to selecting the optimization of ion monitoring (SIM) model selection ion, strengthens antijamming capability, reduces detectability and improves quantitative reappearance.
Embodiment seven:
Derivative reaction is generally 70 ℃ of heating 45min down, microwave heating derivative reaction by experiment, it is constant to fix other condition, change microwave heating time, derive 2,3,4,5,6,7 respectively, 8min, the result is under the high-grade 800W condition of microwave, derivatization 5min, the relative content maximum of melamine-derived product, peak area are with under 70 ℃, and 45min is suitable substantially in heating.The optimum selected time 5min that derives has shortened the reaction time greatly.
Embodiment eight:
Referring to accompanying drawing 1 is full scan (SCAN) mass spectrogram of melamine.Select ion monitoring (SIM) to be often used in and reduce detectability and improve quantitative reappearance.The ion of SIM is selected effectively to eliminate complicated substrate and is disturbed, the characteristic ion of melamine is 99,171,197,285,327,342, through analysis to milk powder mark-on sample, contain 99,171 fragment ions in the sample substrate, quantitative measurement is disturbed big, the characteristic ion of final selected melamine is 197,285,327,342, adopt the incompatible quantitative and conclusive evidence of above-mentioned ion set, accompanying drawing 2 is for adding 1.0mg/kg melamine milk powder sample SIM chromatogram, and the retention time of melamine is 6.985min.
Embodiment nine:
The relative abundance of the retention time of melamine, quota ion, qualitative ion and quota ion and qualitative ion sees Table 1.
Table 1: the relative abundance of the retention time of melamine, quota ion, qualitative ion and quota ion and qualitative ion
| Compound | Retention time (min) | Quota ion (m/z) | Qualitative ion 1 (m/z) | Qualitative ion 2 (m/z) | Qualitative ion 3 (m/z) |
| Melamine (Melamine) | 6.98 | 327(100) | 342(50) | 285(10) | 197(7) |
Qualitative foundation: when carrying out sample determination, if the mass chromatography peak retention time consistent with standard operation solution (variation range is within ± 0.5%) in the sample; And in the sample mass spectrogram after background correction, selected ion all occurs, and the relative abundance of selected ion relative abundance and the suitable standard solution of concentration is more or less the same in 20%, but then has melamine in the judgement sample.
Quantivative approach: external standard method is quantitative, adopts this area routine techniques as can be known.
Embodiment ten:
Preparation melamine standard series working solution, melamine concentration is between the 0.025-2.50 μ g/mL, the machine test is gone up in the back of deriving.By the computed in software range of linearity, related coefficient.Detection limit (LOD) is meant that signal to noise ratio (S/N ratio) (S/N) was greater than 3 o'clock minimal detectable concentration.Linear equation, related coefficient, detection limit see Table 2.
Table 2: the melamine range of linearity, related coefficient, linear equation and detection limit measurement result
| Compound | The range of linearity (μ g/mL) | Related coefficient | Linear equation | Detection limit (mg/kg) |
| Melamine (Melamine) | 0.025-2.50 | 0.9991 | 1.564×10 6X-5.989×10 4 | 0.02 |
Embodiment 11:
With different classes of milk powder is sample, do the interpolation test of three levels, be respectively 0.25mg/kg, 0.5mg/kg, 1.0mg/kg, each interpolation level repeats 6 times, the method recovery and relative standard deviation meet the requirement of domestic and international related standards and rules, see Table 3.
Table 3: milk powder sample melamine adds determination of recovery rates result (n=6)
| Interpolation level (mg/kg) | Measured value (mg/kg) | Mean value (mg/kg) | Average recovery rate (%) | Relative standard deviation (%) |
| 0.25 | 0.28、0.27、0.25、0.24、0.28、0.28 | 0.27 | 107 | 6.6 |
| 0.5 | 0.44、0.44、0.47、0.45、0.50、0.48 | 0.46 | 93 | 5.2 |
| 1.0 | 1.08、0.91、0.89、0.98、0.83、0.93 | 0.94 | 94 | 9.2 |
Claims (4)
1, the method for melamine in a kind of fast measuring milk powder, milk powder are after pre-treatment, and gas chromatography-mass spectrum is measured, and select the ion monitoring pattern, and external standard method is quantitative, it is characterized in that, described pre-treatment step is as follows:
Take by weighing 0.50 gram milk powder sample in 15mL tool vice polypropylene graduated centrifuge tube, add 10mL acetate-acetonitrile-aqueous solution jolting, acetate-acetonitrile-aqueous solution is according to volume ratio preparation in 1: 70: 29, ultrasonic Extraction 10-30min, the centrifugal 5-10min of 4500r/min, filter after solid-phase extraction column activates with the methyl alcohol of 3-5mL successively, the 3-5mL water balance, accurately pipette 4-6mL filtrate upper prop, use the 0.1-0.3mol/L hydrochloric acid solution of 3-5mL successively, the methyl alcohol drip washing of 3-5mL, with rubber pipette bulb pillar is dried up, with 3-5mL5% ammoniacal liquor methanol-eluted fractions, collect eluent with 5mL scale test tube, 45-75 ℃ of nitrogen blow be concentrated into do after, add 200 μ L pyridines and 200 μ L derivatization reagents, with the sealing of glass ground joint plug, be transferred in the sample bottle behind the heated by microwave derivative reaction, air feed phase chromatography-mass spectroscopy is measured.
2, according to the method for melamine in the mensuration milk powder of claim 1, it is characterized in that, described ion monitoring pattern, the characteristic ion of selected melamine is the incompatible quantitative and conclusive evidences of 197,285,327,342 ion set.
3, according to the method for melamine in the mensuration milk powder of claim 1, it is characterized in that, described microwave heating derivative reaction under the high-grade 800W condition of microwave, derivatization 2-8min.
4, according to the method for melamine in the mensuration milk powder of claim 3, it is characterized in that described derivatization 5min.
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