CN105277634A - Method for simultaneously and quantitatively detecting contents of melamine and cyanuric acid and application thereof - Google Patents
Method for simultaneously and quantitatively detecting contents of melamine and cyanuric acid and application thereof Download PDFInfo
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Abstract
The invention discloses a method for simultaneously and quantitatively detecting contents of melamine and cyanuric acid. By discoverying a sovlent capable of greatly codissolving melamine and cyanuric acid under the condtion of non-strong acid and strong base, detection results are truthful and accurate. According to the method, preparation of a sample is simple to operate, good mutual solution of melamine and cyanuric acid can be realized, and an experimental base is provided for accurate measurement of contents of melamine and cyanuric acid. An instrument used in the method is a high performance liquid chromatograph (HPLC). The equipment is cheap and easily available in comparison with equipment used in liquid chromatography tandem mass spectrometry. In addition, a solvent and a mobile phase used in the invention cause little damage to a chromatographic column of the instrument. The detection process is rapid and economic. The method is applied in simultaneously and quantitatively detecting contents of melamine and cyanuric acid, and obtained detection results are accurate and credible. The method is suitable for large-scale sample detection and is also suitable for various samples which require detection of protein content.
Description
Technical field
The present invention relates to field of food detection, particularly relate to a kind of to melamine and the simultaneous quantitative determination of cyanuric acid content and the application of this method.
Background technology
Melamine (Melamine) is commonly called as extract of protein, it is a kind of triazines nitrogen heterocyclic ring organic compound, common industrial chemicals, can be used for synthetic plastic, coating, the agent of yarn fabric anti-folding anti-shrinking, heat-resisting fancy veneer, building paper, grey leather tans skin agent etc.Melamine is toxic to the urinary system of human body (especially infant), reproductive system, even can cause death, therefore, St Foods Q Supervision & Testing Center explicitly points out, melamine belongs to industrial chemicals, does not allow to add in food.
Protein is one of the activity of sustaining life nutriment needing most absorption, and primarily of amino acid composition, its nitrogen content is generally no more than 30%.Protein detection method employing " Kjeldahl's method " that present stage is general estimates protein content by measuring nitrogen content.So some illegal businessmans are under the ordering about of interests, and in order to make protein content food not up to standard be tested by protein content, just adding nitrogen content in food is that the melamine of 66.7% is to increase the nitrogen content in food.
Cyanuric acid (Cyanuricacid) is also a kind of triazines nitrogen heterocyclic ring organic compound, is also conventional industrial chemicals, is mainly used as composition or the raw material of herbicide, germifuge, bleaching agent etc.Because cyanuric acid can be digested by ruminant animals, therefore also extensively added in animal feed.Milk cattle fooder is added with the feed of cyanuric acid or sprayed the green grass of cyanuric acid herbicide, will contain cyanuric acid in oozy milk.At present in feed for pet and the milk just extruded, cyanuric acid detected.
Research shows, the acute toxicity of independent absorption cyanuric acid or melamine is very low, but both taking in simultaneously than independent a pair renal function of both absorptions and the damage of liver organization much serious, this is because melamine and cyanuric acid can generate by intermolecular ydrogen bonding the reticulate texture crystal being insoluble in water, melamine can also be combined the melamine cyanurate compound generating indissoluble in human body with cyanuric acid, form calculus blocking renal tubule, induced renal damage, causes kidney failure.Therefore in food, the detection of melamine and cyanuric acid content has important practical significance.
Owing to easily generating precipitation when melamine and cyanuric acid coexist, if solvent improper use in the process both detecting at the same time, both are combined into precipitation, the testing result finally obtained is both surplus in sample after precipitation, but not real content, testing result substantial deviation actual value, causes testing result unreliable.At present, the method mainly liquid chromatography tandem mass spectrometry of Simultaneously test melamine and cyanuric acid content, the solvent of this method and mobile phase to the chromatographic column damage of instrument very seriously, substantially reduce the serviceable life of chromatographic column; In addition the instrument and equipment cost used of liquid chromatography tandem mass spectrometry is high, and the condition detected is very large to instrument injury, adds inspection cost, is unfavorable for extensive sample tests; In addition, this method Sample Preparation Procedure is loaded down with trivial details, is also easy to the precipitation causing melamine and cyanuric acid, and testing process difficulty is increased.
Therefore, explore as early as possible and a kind ofly method that is accurate, easy, that simultaneously detect melamine and cyanuric acid content can become the difficult problem that those skilled in the art need to capture.
Summary of the invention
Fundamental purpose of the present invention is the technological deficiency for existing in prior art, provides a kind of easy and simple to handle, result can detect the method for melamine and cyanuric acid by simultaneous quantitative accurately and reliably.
The technical scheme adopted for realizing object of the present invention is:
Simultaneous quantitative detects the method for melamine and cyanuric acid content in sample, comprises the following steps:
(1) preparing standard solution: prepare multiple melamine concentration and cyanuric acid CONCENTRATION DISTRIBUTION at the melamine standard solution of 0-120 μ g/mL and cyanuric acid standard solution with solvent respectively; Wherein, described solvent is the trichloroacetic acid solution of 1% (quality volumn concentration, w/v, g/100mL) or aqueous hydrochloric acid solution and acetonitrile mixed preparing; Or the sodium hydrate aqueous solution of 10mM or potassium hydroxide aqueous solution and acetonitrile mixed preparing;
(2) with the peak area of each standard solution in high performance liquid chromatography (HPLC) detecting step (1), UV detect wavelength is 213nm;
(3) with the concentration of standard solution for horizontal ordinate, with the peak area of correspondence for ordinate, draw out melamine typical curve and cyanuric acid typical curve respectively, and obtain corresponding typical curve equation;
(4) by solvent dilution described in sample step (1) or be dissolved into sample solution, detect the peak area of sample solution with HPLC after, peak area numerical value is brought in the typical curve equation of the correspondence that step (3) obtains, namely obtain the content of melamine and cyanuric acid in sample.
In described solvent, the trichloroacetic acid solution of 1% (quality volumn concentration, w/v, g/100mL) or the volume ratio of aqueous hydrochloric acid solution and acetonitrile are (2.5-3.5): 1; Preferably, in described solvent, trichloroacetic acid solution or aqueous hydrochloric acid solution are 3:1 with the mixed volume ratio of acetonitrile.
In described solvent, the sodium hydrate aqueous solution of 10mM or the volume ratio of potassium hydroxide aqueous solution and acetonitrile are (0.8-1.2): 1; Preferably, in described solvent, sodium hydrate aqueous solution or potassium hydroxide aqueous solution are preferably 1:1 with the mixed volume ratio of acetonitrile.
The mobile phase of high performance liquid chromatography is the mixed liquor of the ammonium acetate solution of 10mM and acetonitrile 10:90-20:80 mixed preparing by volume in step (2); In described mobile phase, ammonium acetate solution and the mixed volume of acetonitrile are than preferred 15:85.
The concentration of described melamine standard solution and cyanuric acid standard solution is followed successively by 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 50 μ g/mL, 100 μ g/mL.
The flow velocity of described mobile phase is 1.0mL/min, and the chromatographic column column temperature of described high performance liquid chromatography is 35 DEG C.
The present invention also provides a kind of simultaneous quantitative to detect the method for melamine and cyanuric acid content in food, said method simultaneous quantitative is utilized to detect the content of melamine and cyanuric acid in food, only that the sample in step (4) is replaced to described food, and with dissolution with solvents described in step (1) or be diluted to wherein melamine concentration or cyanuric acid concentration in 0-120 μ g/mL, filter after abundant mixing, obtain food solution, detect the peak area of food solution with HPLC after, peak area numerical value is brought in corresponding typical curve equation, namely the content of melamine and cyanuric acid in food is obtained.
Described food comprises dairy produce, containing one or more in dairy produce and bean product.
Described dairy produce comprises milk, Yoghourt, milk powder, cheese; The described dairy produce that contains comprises sour milk beverage, toffee, cream, milk tea, milk tea powder, milk shake, chocolate, biscuit, cake.
Described bean product comprise soymilk, soymilk powder, soya-bean milk, bean curd.
Provided by the invention simultaneously simple to the preparation manipulation of sample in melamine and cyanuric acid quantitative detecting method, the good molten altogether of melamine and cyanuric acid can be realized, for the content value of both Accurate Determinings provides the foundation; The instrument used in this method is high performance liquid chromatography (HPLC), this equipment is cheap compared with the equipment used in liquid chromatography tandem mass spectrometry, it is a lot of to be easy to get, and the solvent used and mobile phase very little to the injury of the chromatographic column of instrument, the serviceable life of chromatographic column is 2-3 times of liquid chromatography tandem mass spectrometry, and testing process is quick, economical.The testing result that the inventive method obtains is accurately credible, is not only applicable to large-scale sample detection, is also widely used in all kinds of sample simultaneously.
Accompanying drawing explanation
Fig. 1 is that melamine is dissolved in the canonical plotting drawn out in solvent b;
Fig. 2 is that cyanuric acid is dissolved in the canonical plotting drawn out in solvent b;
Fig. 3 is that melamine is dissolved in the canonical plotting drawn out in solvent c;
Fig. 4 is that cyanuric acid is dissolved in the canonical plotting drawn out in solvent c;
Fig. 5 is the blank spectrogram using solvent b to obtain by the inventive method;
Fig. 6 is the test sample spectrogram using solvent b to obtain by the inventive method
Fig. 7 is the blank spectrogram using solvent c to obtain by the inventive method;
Fig. 8 is the test sample spectrogram using solvent c to obtain by the inventive method.
Embodiment
The present invention passes through great many of experiments, find melamine can in acetonitrile excellent dissolution, cyanuric acid also can in acetonitrile excellent dissolution, but the acetonitrile solution mixing of the acetonitrile solution of melamine and cyanuric acid be there will be a large amount of precipitations, this is because melamine and cyanuric acid reaction generate the melamine cyanurate compound being insoluble in acetonitrile.On the basis that this finds, inventor explores a kind of solvent can realize the good molten altogether of melamine and cyanuric acid, and explore and be a kind ofly applicable to the mobile phase that high performance liquid chromatography detects these two kinds of materials simultaneously, thus establish a kind of method utilizing HPLC simultaneous quantitative to detect melamine and cyanuric acid content, and this method is applied to all kinds of quantitative detection containing melamine and cyanuric acid in protein food.
Below in conjunction with the drawings and specific embodiments, further illustrate content of the present invention, and the present invention is further elaborated, but these embodiments there is any restriction to the present invention absolutely not.Those skilled in the art under the enlightenment of this instructions to the embodiment of the present invention in any variation of doing all will belong in the scope of claims of the present invention.
In embodiment, method therefor is conventional method if no special instructions.If no special instructions, in each embodiment the material of same names or reagent content identical.
Embodiment 1, simultaneous quantitative detect the content of melamine and cyanuric acid
One, experimental apparatus and material
High performance liquid chromatography: water generation company of the U.S., Waters2695 type, 2489 UV-detector, Empower II workstation; Chromatographic column: HILICSilica (150 × 4.6mmid.5 μm);
Melamine standard items: purchased from German Dr.Ehrensorfer company, purity 99.0%;
Cyanuric acid standard items: purchased from German Dr.Ehrensorfer company, purity 99.5%;
Acetonitrile, ammonium acetate: purchased from American MERCK company, chromatographically pure;
Experimental water is ultrapure water;
All the other reagent are experiment conventional reagent, analyze pure.
Two, the preparation of mobile phase, solvent and each concentration melamine solution, cyanuric acid solution
1, the preparation of mobile phase
Take ammonium acetate 2.312g (30mmol), adding ultrapure water to 3L makes ammonium acetate dissolve completely, then adds the acetonitrile of 17L wherein, namely obtains acetonitrile-10mM ammonium acetate solution, using this solution as mobile phase after mixing.
2, the preparation of solvent
Solvent a: be the mobile phase in two 1;
Solvent b: take 6g trichloroacetic acid and add ultrapure water to 600mL, stirring makes trichloroacetic acid dissolve completely, then adds the acetonitrile of 200mL wherein, namely obtains acetonitrile-1% (quality volumn concentration after mixing, w/v, g/100mL) trichloroacetic acid solution;
Solvent c: take 0.2g NaOH (NaOH, 5mmol) and add ultrapure water to 500mL, stirs and NaOH is dissolved completely, then add the acetonitrile of 500mL wherein, namely obtain acetonitrile-10mM sodium hydrate aqueous solution after mixing.
3, the preparation of each concentration melamine solution and cyanuric acid solution
1) take 1g melamine, 1g cyanuric acid respectively, then be added in the solvent b of 1mL respectively, the ultrasonic solute that makes dissolves the formation melamine mother liquor of 1g/mL and the cyanuric acid mother liquor of 1g/mL completely;
2) with solvent b respectively by step 1) the melamine mother liquor of 1g/mL that obtains and the cyanuric acid mother liquor of 1g/mL be diluted to the concentration in table 1 and number (see table 1), namely obtains melamine solution and the cyanuric acid solution of a series of variable concentrations.
Repeat step 1) and 2), be only that solvent b is replaced to solvent c or solvent a, the concentration of the melamine solution obtained and cyanuric acid solution and numbering are in table 1.
The concentration table of table 1 melamine solution and cyanuric acid solution
Three, the drafting of typical curve
For melamine, when melamine concentration is in the solution less than 120 below μ g/mL, under identical HPLC testing conditions, the peak area of melamine and melamine concentration is in the solution linear.According to this principle, each mixed standard solution b3-b8 is mixed to get with corresponding solution b23-b28 respectively with the solution b13-b18 prepared in the present embodiment, as solution b13 and solution b23 is mixed to get mixed standard solution b3 ..., solution b18 and solution b28 is mixed to get mixed standard solution b8; Solution c13-c18 is mixed to get each mixed standard solution c3-c8 with corresponding solution c23-c28 respectively, as solution c13 and solution c23 is mixed to get mixed standard solution c3 ..., solution c18 and solution c28 is mixed to get mixed standard solution c8.HPLC is utilized to detect the peak area (blank is respectively above each solution corresponding solvent b or solvent c) of these mixed standard solutions (mixed standard solution b3-b8 and mixed standard solution c3-c8) respectively.Wherein, mobile phase adopts 1 mobile phase prepared of in the present embodiment two, and the column temperature of flow velocity 1.0mL/min, HPLC chromatographic column is 35 DEG C, and determined wavelength is 213nm.Then with solute (melamine) concentration (μ g/mL) for horizontal ordinate, peak area is that ordinate is made the typical curve of melamine respectively in solvent b, solvent c and simulates typical curve equation (see table 2) corresponding to every bar typical curve (see Fig. 1 and Fig. 3).Cyanuric acid is also drawn according to the method described above and is obtained cyanuric acid at different solvents standard curve, sees Fig. 2 and Fig. 4, and simulates corresponding typical curve equation, in table 2.
The typical curve equation of melamine, cyanuric acid in table 2 different solvents
Four, the mensuration of melamine, cyanuric acid content
Take food product solvent b (or solvent c) to dissolve, filter, get supernatant (instant food solution), the peak area of melamine in (in testing conditions and three of the present embodiment, testing conditions is consistent) food solution and cyanuric acid is detected with HPLC, the approximate content obtaining melamine and cyanuric acid in food solution is estimated by peak area, then by food solution dilution to wherein melamine and cyanuric acid content at 120 μ g/mL (upper limit of the range of linearity) below, obtain food solution to be measured, the peak area of melamine in (in testing conditions and three of the present embodiment, testing conditions is consistent) food solution to be measured and cyanuric acid is detected with HPLC, again this peak area is brought in typical curve equation corresponding to solvent b in table 2 (or solvent c), calculate the content (μ g/mL) of melamine and cyanuric acid, .Wherein, blank is used solvent.
Five, the inspection of the inventive method repeatability, validity
In order to check and verify the validity to melamine and cyanuric acid Simultaneously test of the inventive method, following repeated experiment and validity experiment are carried out.
1, the inspection of the inventive method validity
The solvent used to check the inventive method, to the Detection results of melamine and cyanuric acid content, has done following experiment.Get two kinds of solution equal-volumes mixing that the identical but solute of solvent in table 1 is different, HPLC is utilized to detect the peak area of (in testing conditions and three of the present embodiment, testing conditions is consistent) mixed solution, typical curve is utilized to calculate the content (experiment value) of wherein melamine and cyanuric acid, and contrast with the theoretical value of solutes content in mixed solution, the results are shown in Table 3 and table 4.
The result 1 of table 3 the inventive method validity
As can be seen from Table 3, in the identical situation of other experiment conditions, solvent becomes the whether accurate key of experimental result.Find in experimentation, melamine and cyanuric acid precipitate in solvent a, due to the 1:1 reaction in molar ratio of melamine and cyanuric acid, therefore in experiment value cyanuric acid concentration be 0 result show that cyanuric acid precipitates completely, not containing cyanuric acid in mixed solution; That is, after going to dissolve melamine and cyanuric acid with solvent a, the content of solute after the testing result obtained is actually solute precipitates, in solution, is remained, instead of the real content of solute, with legitimate reading substantial deviation.And melamine and cyanuric acid can be molten altogether well in solvent b and solvent c, do not precipitate, thus make testing result true and accurate, its relative error is all less than 5%, and (constant for animal doctor field of veterinary HPLC method measures, relative error is within ± 10%, be trust data), the solvent that explanation solvent b and solvent c detects in the method for melamine and cyanuric acid as simultaneous quantitative of the present invention is feasible, and experimental result is true and reliable.
This is because melamine and cyanuric acid are dissolved in acetonitrile solution completely, after adding trichloroacetic acid (solvent b) or NaOH (solvent c), prevent the combination of the two intermolecular ydrogen bonding, avoid the two generation precipitation that reacts that coexists.
In addition, also by the melamine solution of lower solute solubility and the mixing of cyanuric acid solution, detect according to above-mentioned same procedure, verify the validity of the inventive method, the results are shown in Table 4.
The result 2 of table 4 the inventive method validity
Can be drawn by the result in table 4, the experimental result error using solvent a to obtain is too large, and be not suitable for the content detecting melamine in sample and cyanuric acid, reason is with the analysis of experimental result in his-and-hers watches 3.When using solvent b and solvent c, the general trend of experimental result be the validity that can reflect the inventive method (for the determination of trace of animal doctor field of veterinary HPLC method, relative error is within ± 25%, be trust data), but the error of individual data is slightly large, this may be because the concentration of solute is low, under this experiment condition, the interference of other factors becomes and can not ignore, so cause experimental result to occur deviation, with solvent b and solvent c, as simultaneous quantitative of the present invention, the solvent detected in the method for melamine and cyanuric acid is feasible on the whole, and experimental result is true and reliable.
The experimental result of conclusive table 3 and table 4, can show that simultaneous quantitative of the present invention detects in the method for melamine and cyanuric acid content uses solvent b or solvent c to be realize key of the present invention as solvent, exactly because also explore this two kinds of solvents, demonstrate the method for melamine and cyanuric acid content of validity the present invention detects to(for) simultaneous quantitative.Therefore follow-up experiment only uses solvent b or solvent c as the solvent of melamine solution and cyanuric acid solution.
2, the inspection of the inventive method repeatability
In order to check the reappearance of the inventive method, do following experiment.Get solution b18 and each 1mL of solution b28, solution c18 and each 1mL of solution c28, mix the rear HPLC that utilizes respectively and detect (in testing conditions and three of the present embodiment, testing conditions is consistent) mixed solution 6 times (all the other are consistent with the validity step of inspection the inventive method), calculate the relative standard deviation (RSD) of 6 peak areas, the results are shown in Table 5.
The result of table 5 the inventive method repeatability
As can be seen from the experimental result of table 5, when selecting solvent b or solvent c to realize the inventive method, standard deviation is all less than 5%, illustrate that the inventive method can be used in simultaneous quantitative and detects the content of melamine and cyanuric acid and have reproducibility, the precision of the inventive method is high, for the accuracy of experimental result lays the foundation.
Six, the inventive method detects the determination of lower limit
In the present embodiment five 1 table 4 in found out that, when the concentration of melamine and cyanuric acid is too low, experimental result deviation is comparatively large, detect lower limit because this has been following experiment to what obtain the inventive method.Getting solution b11 and solution b21, to be diluted to solute concentration with solvent b be respectively 0.3 μ g/mL, 0.2 μ g/mL, 0.1 μ g/mL and 0.05 μ g/mL, the solution of each concentration after utilizing HPLC detection (in testing conditions and three of the present embodiment, testing conditions is consistent) to dilute, calculate the content of melamine and cyanuric acid, and using concentration during signal to noise ratio (S/N ratio)=3 as minimum detectability.Experimental result shows that the inventive method is 0.2 μ g/mL to the minimum detectability of melamine, is 0.5 μ g/mL to the minimum detectability of cyanuric acid.
Seven, the determination of the inventive method solvent kind and proportioning
1, the determination of solvent kind
According to the literature found at present, in most research, all adopt acetonitrile solution as solvent, and through checking, in acetonitrile solution, melamine and cyanuric acid can generate precipitation, make measurement result substantial deviation true value.Separately have in part research and adopt formic acid-acetonitrile solution as solvent, its validity is also poor, because the mensuration of formic acid summit interference cyanuric acid wherein.
The present invention utilizes above experimental technique to also compares the common molten situation of melamine and cyanuric acid in the common acid conditions such as acetonitrile-aqueous hydrochloric acid solution, acetonitrile-aqueous sulfuric acid, acetonitrile-aqueous acetic acid, found that, in acetonitrile-aqueous hydrochloric acid solution, the two can realize good molten altogether, and there will be protein in acetonitrile-aqueous sulfuric acid by the problem of " carbonization ", interference measurement result, in acetonitrile-aqueous acetic acid, two kinds of solutes cannot coexist, can generate precipitation.
In addition, the present invention also compares the common molten situation of melamine and cyanuric acid in the common alkali conditions such as acetonitrile-potassium hydroxide aqueous solution, acetonitrile-concentrated ammonia solution, acetonitrile-triethylamine aqueous solution, find through research: acetonitrile-potassium hydroxide aqueous solution also can stop melamine and cyanuric acid to generate precipitation, but in the alkalescent solvent that acetonitrile-concentrated ammonia solution, acetonitrile-triethylamine aqueous solution etc. are conventional, two kinds of solutes can react and generate precipitation, cause measurement result inaccurate.
In sum, only in part strong acid, strong alkali environment, just really can realize coexisting, so the present invention finally selects trichloroacetic acid, hydrochloric acid, NaOH or potassium hydroxide as the solvent coordinated with acetonitrile of melamine and cyanuric acid; The solvent that wherein hydrochloric acid coordinates with acetonitrile is acetonitrile-1% (quality volumn concentration, w/v, g/100mL) aqueous hydrochloric acid solution, potassium hydroxide and NaOH similar, use acetonitrile-10mM potassium hydroxide aqueous solution.
2, the determination of solvent burden ratio
By the compound method of solvent b and solvent c in embodiment 1, only adjust solvent kind and proportioning, obtain a series of solvent, as shown in table 6.And then according to the validity of solvent in the step check table 6 of Part V in embodiment 1, result is still in table 6.
The validation verification result of table 6 different solvents
The data of table 6 show, acid causes excessive acid in solution with the too high meeting of proportioning of acetonitrile, thus causes melamine measurement result on the low side; Too low meeting causes cyanuric acid and melamine to coexist completely, can react and generate precipitation, thus cause the two assay result all on the low side, the too high meeting of proportioning of alkali and acetonitrile causes base excess in solution, thus cause cyanuric acid measurement result on the low side, too low meeting causes cyanuric acid and melamine to coexist completely, can react and generate precipitation, thus causes the two assay result all on the low side.
So the volume ratio that the present invention finally selects trichloroacetic acid or hydrochloric acid to coordinate with acetonitrile is (2.5-3.5): 1; The volume ratio that NaOH or potassium hydroxide coordinate with acetonitrile is (0.8-1.2): 1.
The application of embodiment 2, melamine and cyanuric acid content simultaneous quantitative determination
One, the inventive method is used for the mensuration of melamine and cyanuric acid content in milk powder
Get milk powder (from cattle farm, Beijing self-control milk powder, wherein not containing melamine and cyanuric acid) each 1g of 1g, melamine and cyanuric acid, add solvent b and be dissolved to 100mL, ultrasonic mixing, namely obtain sample mother liquor; Get 1mL sample mother liquor, 500 times are diluted with solvent b, 0.45 μm of filtration, obtain the test sample that melamine and cyanuric acid concentration are 20 μ g/mL, the peak area that (in testing conditions and three of the present embodiment, testing conditions is consistent) tests melamine in sample and cyanuric acid is detected with HPLC, this peak area is brought in typical curve equation corresponding to solvent b in table 2 again, calculate the content (μ g/mL) of melamine and cyanuric acid.Wherein, blank is milk power solution after the dilution not adding melamine and cyanuric acid, and testing result is shown in Fig. 5, Fig. 6 and table 7.
In this way solvent b is wherein replaced to solvent c, obtain Fig. 7, Fig. 8 and table 7.
The content detection result of melamine and cyanuric acid in table 7 milk powder
Result shows, and the simultaneous quantitative that the inventive method may be used for melamine and cyanuric acid content in milk powder detects, and experimental result accurately and reliably.
In addition, the inventive method is also utilized to detect the melamine in the food such as commercially available milk, toffee, Yoghourt, sour milk beverage, cheese and cyanuric acid content, and use UPLC-MASS method to verify simultaneously, the result that its result and the inventive method obtain is consistent, show that the inventive method can be used for detecting the simultaneous quantitative detection of melamine and cyanuric acid content in the food such as dairy produce, flour, feed for pet, do not repeat one by one at this.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. simultaneous quantitative detects the method for melamine and cyanuric acid content in sample, it is characterized in that, comprises the following steps:
(1) preparing standard solution: prepare multiple melamine concentration and cyanuric acid CONCENTRATION DISTRIBUTION at the melamine standard solution of 0-120 μ g/mL and cyanuric acid standard solution with solvent respectively; Wherein, described solvent is the trichloroacetic acid solution of 1% (quality volumn concentration, w/v, g/100mL) or aqueous hydrochloric acid solution and acetonitrile mixed preparing; Or the sodium hydrate aqueous solution of 10mM or potassium hydroxide aqueous solution and acetonitrile mixed preparing;
(2) with the peak area of each standard solution in high performance liquid chromatography (HPLC) detecting step (1), UV detect wavelength is 213nm;
(3) with the concentration of standard solution for horizontal ordinate, with the peak area of correspondence for ordinate, draw out melamine typical curve and cyanuric acid typical curve respectively, and obtain corresponding typical curve equation;
(4) by solvent dilution described in sample step (1) or be dissolved into sample solution, detect the peak area of sample solution with HPLC after, peak area numerical value is brought in the typical curve equation of the correspondence that step (3) obtains, namely obtain the content of melamine and cyanuric acid in sample.
2. method according to claim 1, it is characterized in that, in described solvent, the trichloroacetic acid solution of 1% (quality volumn concentration, w/v, g/100mL) or the volume ratio of aqueous hydrochloric acid solution and acetonitrile are (2.5-3.5): 1; Preferably, in described solvent, trichloroacetic acid solution or aqueous hydrochloric acid solution are 3:1 with the mixed volume ratio of acetonitrile.
3. method according to claim 1, it is characterized in that, in described solvent, the sodium hydrate aqueous solution of 10mM or the volume ratio of potassium hydroxide aqueous solution and acetonitrile are (0.8-1.2): 1; Preferably, in described solvent, sodium hydrate aqueous solution or potassium hydroxide aqueous solution are preferably 1:1 with the mixed volume ratio of acetonitrile.
4. according to the arbitrary described method of claim 1-3, it is characterized in that, the mobile phase of high performance liquid chromatography is the mixed liquor of the ammonium acetate solution of 10mM and acetonitrile 10:90-20:80 mixed preparing by volume in step (2); In described mobile phase, ammonium acetate solution and the mixed volume of acetonitrile are than preferred 15:85.
5. according to the arbitrary described method of claim 1-4, it is characterized in that, the concentration of described melamine standard solution and cyanuric acid standard solution is followed successively by 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 30 μ g/mL, 50 μ g/mL, 100 μ g/mL.
6. according to the arbitrary described method of claim 1-5, it is characterized in that, the flow velocity of described mobile phase is 1.0mL/min, and the chromatographic column column temperature of described high performance liquid chromatography is 35 DEG C.
7. simultaneous quantitative detects the method for melamine and cyanuric acid content in food, it is characterized in that, the arbitrary described method simultaneous quantitative of claim 1-6 is utilized to detect the content of melamine and cyanuric acid in food, only that the sample in step (4) is replaced to described food, and with dissolution with solvents described in step (1) or be diluted to wherein melamine concentration or cyanuric acid concentration in 0-120 μ g/mL, filter after abundant mixing, obtain food solution, detect the peak area of food solution with HPLC after, peak area numerical value is brought in corresponding typical curve equation, namely the content of melamine and cyanuric acid in food is obtained.
8. method according to claim 7, it is characterized in that, described food comprises dairy produce, containing one or more in dairy produce and bean product.
9. method according to claim 8, it is characterized in that, described dairy produce comprises milk, Yoghourt, milk powder, cheese; The described dairy produce that contains comprises sour milk beverage, toffee, cream, milk tea, milk tea powder, milk shake, chocolate, biscuit, cake.
10. method according to claim 8 or claim 9, it is characterized in that, described bean product comprise soymilk, soymilk powder, soya-bean milk, bean curd.
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| CN109470802A (en) * | 2019-01-10 | 2019-03-15 | 通标标准技术服务(上海)有限公司 | A method of melamine phosphate content in measurement textile or foam |
| CN111257460A (en) * | 2020-02-25 | 2020-06-09 | 中国水产科学研究院黄海水产研究所 | Detection method of triazine herbicide and metabolite thereof in shellfish |
| CN111721864A (en) * | 2020-06-16 | 2020-09-29 | 山东省食品药品检验研究院 | Method for rapidly determining non-edible substance trithiocyanuric acid trisodium salt in wheat flour and additives thereof by high performance liquid chromatography |
| CN111721863A (en) * | 2020-06-16 | 2020-09-29 | 山东省食品药品检验研究院 | Method for measuring non-edible substance trithiocyanuric acid trisodium salt in wheat flour and additives thereof by ultra-high performance liquid chromatography-tandem mass spectrometry |
| CN112379027A (en) * | 2020-10-28 | 2021-02-19 | 上海国齐检测技术有限公司 | Method for detecting melamine in soil |
| CN114720628A (en) * | 2022-04-07 | 2022-07-08 | 山东泰星新材料股份有限公司 | Method for determining content of free melamine in melamine cyanurate |
| CN116165196A (en) * | 2023-04-13 | 2023-05-26 | 河南心连心化肥检测有限公司 | Method for detecting cyanuric acid in high-purity urea |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101387609A (en) * | 2008-10-20 | 2009-03-18 | 中国农业大学 | Method for rapidly detecting melamine |
| CN101446574A (en) * | 2008-10-10 | 2009-06-03 | 上海市徐汇区中心医院 | LC-MS quantitative detection method for melamine and tricyanic acid in human urine |
-
2015
- 2015-09-30 CN CN201510641556.3A patent/CN105277634A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101446574A (en) * | 2008-10-10 | 2009-06-03 | 上海市徐汇区中心医院 | LC-MS quantitative detection method for melamine and tricyanic acid in human urine |
| CN101387609A (en) * | 2008-10-20 | 2009-03-18 | 中国农业大学 | Method for rapidly detecting melamine |
Non-Patent Citations (7)
| Title |
|---|
| S. A. TITTLEMIER: "Methods for the analysis of melamine and related compounds in foods: a review", 《FOOD ADDITIVES AND CONTAMINANTS》 * |
| S. EHLING ET AL.: "High-performance liquid chromatographic method for the simultaneous detection of the adulteration of cereal flours with melamine and related triazine by-products ammeline, ammelide, and cyanuric acid", 《FOOD ADDITIVES AND CONTAMINANTS》 * |
| 中华人民共和国国家质量监督检验检疫总局 等: "《GB/T 22388-2008 原料乳与乳制品中三聚氰胺检测方法》", 30 November 2008, 中国标准出版社 * |
| 于晓辉 等: "高效液相色谱法同时测定三氯异氰脲酸(粉)中三聚氰酸和三聚氰胺的含量", 《化学分析计量》 * |
| 李锋格 等: "混合型固相萃取柱净化-气相色谱-质谱法测定婴幼儿配方奶粉中的三聚氰酸", 《色谱》 * |
| 林晓珊 等: "气相色谱-串联质谱法快速测定乳制品中三聚氰胺及其３种类似物", 《质谱学报》 * |
| 马丽莎 等: "食品中三聚氰胺与三聚氰酸残留检测研究进展", 《中国兽药杂质》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109470802A (en) * | 2019-01-10 | 2019-03-15 | 通标标准技术服务(上海)有限公司 | A method of melamine phosphate content in measurement textile or foam |
| CN111257460A (en) * | 2020-02-25 | 2020-06-09 | 中国水产科学研究院黄海水产研究所 | Detection method of triazine herbicide and metabolite thereof in shellfish |
| CN111721864A (en) * | 2020-06-16 | 2020-09-29 | 山东省食品药品检验研究院 | Method for rapidly determining non-edible substance trithiocyanuric acid trisodium salt in wheat flour and additives thereof by high performance liquid chromatography |
| CN111721863A (en) * | 2020-06-16 | 2020-09-29 | 山东省食品药品检验研究院 | Method for measuring non-edible substance trithiocyanuric acid trisodium salt in wheat flour and additives thereof by ultra-high performance liquid chromatography-tandem mass spectrometry |
| CN111721864B (en) * | 2020-06-16 | 2022-04-22 | 山东省食品药品检验研究院 | Method for rapidly determining non-edible substance trithiocyanuric acid trisodium salt in wheat flour and additives thereof by high performance liquid chromatography |
| CN112379027A (en) * | 2020-10-28 | 2021-02-19 | 上海国齐检测技术有限公司 | Method for detecting melamine in soil |
| CN114720628A (en) * | 2022-04-07 | 2022-07-08 | 山东泰星新材料股份有限公司 | Method for determining content of free melamine in melamine cyanurate |
| CN116165196A (en) * | 2023-04-13 | 2023-05-26 | 河南心连心化肥检测有限公司 | Method for detecting cyanuric acid in high-purity urea |
| CN116165196B (en) * | 2023-04-13 | 2023-08-29 | 河南心连心化肥检测有限公司 | Method for detecting cyanuric acid in high-purity urea |
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