CN103217498A - Method for detecting dicyandiamide in milk powder with LC-MS (liquid chromatography/mass spectrometry) and sample preparation method - Google Patents
Method for detecting dicyandiamide in milk powder with LC-MS (liquid chromatography/mass spectrometry) and sample preparation method Download PDFInfo
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Abstract
The invention provides a sample preparation method for detecting dicyandiamide in milk powder with LC-MS (liquid chromatography/mass spectrometry). The sample preparation method comprises the following steps of: dissolving a sample in water, adding an organic extracting solution to the dissolved sample to extract, adding MgSO4 and NaCl for layering the organic extracting solution and a water phase, centrifuging the solution to obtain first liquid supernatant, adding an adsorbing material to the first liquid supernatant for purifying, centrifuging the purified first liquid supernatant to obtain second liquid supernatant, and concentrating, re-dissolving and filtering the second liquid supernatant to obtain a sample to be detected. A method for detecting dicyandiamide in milk powder with LC-MS comprises the steps of preparation of the sample and detection on dicyandiamide in milk powder by an ultra-high performance liquid chromatography-triple four-level rod mass spectrum combining instrument. The sample preparation method suitable for LC-MS detection is capable of removing interference molecules through extraction and purification, and thus the accuracy of sample detection is higher.
Description
Technical field
The invention belongs to the food inspection field, be specifically related to the method for dicyandiamide in a kind of fast detecting milk powder, and liquid-matter coupling detects the sample-pretreating method of dicyandiamide.
Background technology
Dicyandiamide (DCD) has another name called dicyanodiamine, cyanamid dimerization, and chemical formula is C
2H
4N
4, be a kind of raw material of industry, the adjuvant of Chang Zuowei compound fertilizer uses, and with the activity of control nitrifier, regulates the conversion rate of nitrogenous fertilizer in soil, reduces the loss of ammonium nitrogen, reaches the purpose that improves the fertilizer service efficiency.In January, 2013, the permanent natural group of New Zealand dairy industry giant is produced in the part milk powder of its production contains dicyandiamide, and its cause is because the New Zealand dairy farmer has used the chemical fertilizer that contains dicyandiamide, thereby it is residual to cause having produced dicyandiamide in the milk powder.Dicyandiamide is a raw material of producing melamine, belong to the high nitrogen material of non-albumen equally, its security does not obtain assessment as yet, and in addition, dicyandiamide is as the dimer of cyanamide, it is similar to mouse poisoning symptom and cyanamide tripolymer melamine, consistent poisoning symptom appears in the mouse after the dicyandiamide of 13000mg/kg dosage and the melamine administration, all occurs uneasyly, jumps, be short of breath, just dead in dozens of minutes subsequently.In view of it may produce harm to the infant as melamine, Government Of New Zealand forbids selling the milk system product that contains dicyandiamide.China is the main body entrance state of New Zealand's milk powder, at present about on the market 40% milk powder is from New Zealand, and this time incident has caused people's showing great attention to and query milk powder safety once again.
Chinese patent literature CN102419354A discloses the general method for quick of micromolecule objectionable impurities in a kind of liquid milk, and specifically discloses the method that adopts Ultra Performance Liquid Chromatography and triple level Four bar mass spectrometry to measure micromolecule objectionable impurities wherein; Described method is applicable to micromolecule objectionable impuritiess such as detecting melamine in fluid milk, dicyandiamide.Chinese patent CN102435699A discloses the method for melamine in a kind of liquid chromatography tandem mass spectrum fast detecting breast and the dairy products, and specifically disclose in dairy products to be measured and to have marked in adding, extract melamine with acetonitrile, with the step of melamine in qualitative, the quantitative analysis of forward chromatographic column tandem mass spectrum MRM, the measurement dairy products.As seen, when detecting in breast or the dairy produce micromolecule objectionable impurities such as dicyandiamide or melamine at present, the mode of liquid phase-mass spectrum logotype becomes detection mode the most fast and accurately.
But in practical study, find; when detecting dicyandiamide in the milk powder, above-mentioned mode with liquid phase-mass spectrometry finds; because the different in kind of milk powder is in liquid milk or dairy products; when make adopting said method carry out that dicyandiamide detects in the milk powder to detect effect unsatisfactory; the pre-treating method of the sample in most importantly above-mentioned two kinds of methods also is not suitable for the detection of dicyandiamide in the milk powder; no matter be Chinese patent CN102419354A mention extract the pre-treatment mode that melamine is purified with what protective agent and acetonitrile-alcohol mixeding liquid carried out mentioning among the mode of purification processes or the Chinese patent CN102435699A with acetonitrile; the sample solution that obtains (being testing sample); wherein all inevitably still there are a large amount of protein molecules; fat molecule and the disturbing molecule that some are close with the dicyandiamide molecular weight; it is little thereby even employing has interference; detection limit is low; highly sensitive; when the Ultra Performance Liquid Chromatography of advantages such as favorable reproducibility and the coupling of triple level Four bar are measured above-mentioned liquid to be measured; degree of accuracy is still relatively poor; this mainly be since milk powder in preparation during testing sample; generally all be simply to separate with water-soluble; cause a lot of protein molecules and fat molecule to utilize acetonitrile to purify because of effectively dissolving; also just directly influenced the follow-up sensitivity that detects; therefore; need badly and develop a kind of new sample-pretreating method that is applicable to dicyandiamide fast detecting in the milk powder, especially be applicable to the method for the sample pre-treatments of the efficient and sensible that liquid-matter coupling detects.
Summary of the invention
Problem to be solved by this invention is that the detection to dicyandiamide in the milk powder causes the relatively poor problem of testing result precision owing to the sample pre-treatments mode in the prior art, and then a kind of sample-pretreating method that is applicable to dicyandiamide in the fast detecting milk powder is provided, especially be applicable to the sample-pretreating method that liquid-matter coupling detects;
Second technical matters to be solved by this invention is to provide a kind of method that can fast detecting goes out dicyandiamide in the milk powder.
For solving the problems of the technologies described above, technical scheme provided by the invention is:
A kind of liquid-matter coupling detects the sample-pretreating method of dicyandiamide in the milk powder, and it comprises the following steps:
(1) powdered milk sample is dissolved aqueous solution, and add the extraction that organic extract carries out dicyandiamide;
(2) add MgSO in the mixed liquor after extracting
4Make described organic extract and water layering with NaCl, the centrifugal supernatant of winning;
(3) in described first supernatant, add adsorbent and purify, centrifugal second supernatant that gets;
(4) with described second supernatant through concentrate, redissolution, filtration step, obtain required testing sample.
In the described step (1), the step that described organic extract extracts dicyandiamide is carried out under ultrasound condition, and preferred ultrasonic frequency is 40kHz.
In the described step (1), described organic extract is one or more the potpourri in acetonitrile, acidifying acetonitrile, ammonification acetonitrile, ethyl acetate, methyl alcohol or the ethanol.
The described organic extract concentration height of aqueous solution or variation one or many of volume per sample adds extraction in batches, reaches the purpose of extracting dicyandiamide and get final product, and preferably the volume of described organic extract and the volume ratio of described sample aqueous solution are 5:1.
In the described step (2), described MgSO
4Addition and the quality of described powdered milk sample be 1:1-3:1, the addition of described NaCl and the quality of described powdered milk sample are 0.5:1-1:1.
In the described step (3), described adsorbent is the C18 adsorbent of particle diameter 40-60 μ m.
The addition of described adsorbent and the mass ratio of described powdered milk sample are 0.15:1-0.3:1.
In the described step (4), described concentration step adopts the mode that dries up with nitrogen to carry out.
Described redissolution step is to adopt the mixed liquor of the aqueous formic acid of acetonitrile and 0.1% to carry out, and the volume ratio of described acetonitrile and described formic acid solution is 3:7.
It is 0.22 μ m filter membrane that the aperture is adopted in described filtration.
Further, the present invention also provides a kind of liquid-matter coupling to detect the method for dicyandiamide in the milk powder, and it comprises the steps:
(a) according to the arbitrary described method of claim 1-9 described milk powder is carried out pre-treatment, obtain required testing sample;
(b) adopt Ultra Performance Liquid Chromatography-triple level Four bar GC-MS that the dicyandiamide that contains in the milk powder is detected.
The testing conditions of high performance liquid chromatography is described in the described step (b):
Chromatographic column: Waters ACQUITY UPLC HSS T32.1 * 150mm, 1.8 μ m;
Column temperature: 30 ℃;
Mobile phase A: 10mmol/LNH
4The Ac aqueous solution, Mobile phase B: acetonitrile;
Flow velocity: 0.25mL/min;
Sample size: 10 μ L;
Type of elution: gradient elution;
The mass spectral testing conditions of described triple level Four bars is:
Mass analyzer: triple level Four bars;
Ion gun: ESI
+
Capillary voltage: 4kV;
Taper hole voltage: 25V;
Ion source temperature: 150 ℃;
Desolventizing temperature: 400 ℃;
Center gas flow velocity: 50L/Hr;
Desolventizing gas flow rate: 800L/Hr;
Collision gas velocity: 0.15mL/min.
The gradient elution program of described high performance liquid chromatography is:
Technique scheme of the present invention has the following advantages compared to existing technology:
(1) sample-pretreating method of the present invention, with organic extract sample is carried out the extraction of dicyandiamide after, with MgSO
4With NaCl sample being carried out layering extracts, utilize the increase of salinity in the water, further reduce dicyandiamide, and then increase its concentration of ordinary dissolution in organic phase in aqueous phase solubleness, it is distributed by liquid liquid and more enter organic phase, the extraction efficiency of maximized increase dicyandiamide; Simultaneously because MgSO
4Solubleness in water is less, and has very strong water absorptivity, and produces spontaneous heating in suction, can further increase the solubleness of dicyandiamide in organic phase, thereby improves extraction efficiency; In addition, by means of MgSO
4With the salting out of NaCl, promote organic phase and water layering, improve separation efficiency; And by to the absorption, separation of extracting back solution, concentrate, the processing of steps such as redissolution, filtration, farthest may disturb the factor that detects the result to handle, further promoted detection accuracy and detection sensitivity;
(2) sample-pretreating method of the present invention, when the quality that takes by weighing powdered milk sample is 1g, described MgSO
4Consumption be 1-3g, and preferred 2g had both avoided MgSO
4Consumption causes suction and exothermic effect more weak very little, does not have the problem that improves extraction efficiency, has also avoided MgSO
4Consumption is too big, will produce the caking of bulky grain shape, causes MgSO
4Disperse inhomogeneously with sample substrate, influence the problem of extraction efficiency; And studies show that, at preferred MgSO
4Under the condition of consumption, the MgSO of dispersion
4Solid particle and milk powder matrix form mixture, and play friction and exothermic effects in the vortex process, have improved extraction efficiency effectively; The consumption of described NaCl is 0.5-1g, and preferred 0.5g, both solved the NaCl consumption very little, cause salting out not obvious, and consumption is excessive, have part NaCl and enter acetonitrile layer, follow-up dry up have in the process part salt out, the excessive concentration of salt can suppress the problem of mass spectrum response signal, and efficient but also guaranteed follow-up detection accuracy has not only guaranteed to saltout;
(3) sample-pretreating method of the present invention, utilize adsorbent C18 that sample is carried out purified treatment, thereby can remove protein molecule, fat molecule that itself contains in the testing sample and the disturbing molecule that some are close with the dicyandiamide molecular weight, simultaneously because C18 is a non-polar adsorbent, and the target molecule dicyandiamide is a polar molecule, use adsorbent C18 that sample is carried out purified treatment, in adsorption sample, in the impurity molecule, avoid absorption target molecule;
(4) sample-pretreating method of the present invention, the mode that described concentration step adopts nitrogen to dry up, not only can carry out concentration to a plurality of samples simultaneously, efficient is higher, and compared to the mode of rotary evaporation, nitrogen dries up and more is applicable to small amount of sample, and avoided sample in transfer waste and the experimental error that causes;
(5) sample-pretreating method of the present invention, described redissolution step adopts the mixed liquor of acetonitrile and 0.1% aqueous formic acid, the volume ratio of preferred acetonitrile and 0.1% aqueous formic acid is 3:7, with this understanding, the liquid that redissolves can avoid object to reduce the recovery because of redissolution not yet in effect effectively with the dissolving of the residue on the glass test tube wall; It is protonated that a spot of formic acid helps dicyandiamide, improves the response of instrument;
(6) sample-pretreating method of the present invention, adopting the aperture is that 0.22 μ m filter membrane filters, and to the damage of chromatogram, also plays the effect that purifies sample to prevent to contain big particle diameter impurity molecule in the sample simultaneously to a certain extent;
(7) the present invention adopts Ultra Performance Liquid Chromatography-GC-MS to detect the dicyandiamide that contains in the described testing sample, this detection method has that interference is little, detection limit is low, the characteristics of highly sensitive, favorable reproducibility, and is suitable for the trace materials dicyandiamide that contains in the milk powder is detected.
Description of drawings
For the easier quilt of content of the present invention is clearly understood, below in conjunction with accompanying drawing, the present invention is further detailed explanation, wherein,
Figure 1A, 1B, 1C are respectively dicyandiamide standard items of the present invention total ion current and two selection ion figure on triple level Four bar mass spectrums;
Fig. 2 is the typical curve of dicyandiamide;
Fig. 3 A, Fig. 3 B are the total ion current of dicyandiamide in the whole milk powder negative sample and select ion figure;
Fig. 4 A, Fig. 4 B are respectively the total ion current of dicyandiamide in the whole milk powder positive and select ion figure;
Fig. 5 A, Fig. 5 B are respectively the total ion current of dicyandiamide in the skimmed milk power positive and select ion figure;
Fig. 6 A, Fig. 6 B are respectively the total ion current of dicyandiamide in the baby milk powder negative sample and select ion figure.
Specific implementation method
Further set forth technical characterstic of the present invention below in conjunction with specific embodiment.
Instrument and reagent:
UPLC-Xevo TQ MS type Ultra Performance Liquid Chromatography-tandem mass spectrometer (U.S. Waters company);
Nitrogen evaporator (U.S. Organomation company)
Miillpore filter (0.22 μ m, U.S. Pall company);
Ultrasonic cleaner (Kunshan, Jiangsu ultrasonic instrument company limited);
Supercentrifuge (German Sigma company);
Waters ACQUITY UPLC HSS T32.1 * 150mm, 1.8 μ m chromatographic columns, U.S. Waters company;
The dicyandiamide standard items are available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute; Ammonium acetate (chromatographic grade, U.S. TEDIA company); Acetonitrile (chromatographically pure, U.S. Fisher company); Anhydrous MgSO
4(analyzing pure), NaCl(analyze pure) available from Chemical Reagent Co., Ltd., Sinopharm Group; The C18 adsorbent is available from Ai Jieer company (Cleanert C18,40-60 μ m); Experimental water is by Milli-Q ultrapure water systems produce;
1. the optimization of mass spectrum condition
Be respectively dicyandiamide standard items of the present invention total ion current and two selection ion figure on triple level Four bar mass spectrums shown in Figure 1A, 1B, the 1C, dicyandiamide parent ion ([M+H] in the milk powder matrix experiment of standard items
+) mass number be 85, the mass number of qualitative daughter ion is respectively 68 and 43, wherein, Figure 1B is depicted as the selection ion figure of daughter ion m/z68, Fig. 1 C is depicted as the selection ion figure of daughter ion m/z43, because the daughter ion of m/z43 has Interference Peaks (shown in Fig. 1 C) at the retention time place, therefore selecting m/z68 is quantitative daughter ion (Figure 1B), increase along with collision energy, the parent ion abundance reduces gradually, daughter ion m/z43, the abundance of m/z68 descends gradually, is that the daughter ion of m/z68 is a quota ion so dicyandiamide is finally selected under the collision energy of 12ev specific charge for use, and specific charge is that the daughter ion of m/z43 is auxiliary qualitative daughter ion under the collision energy of 10ev.
2. typical curve
Get the dicyandiamide standard solution, with acetonitrile and 0.1% aqueous formic acid (3:7, v/v) be solvent, stepwise dilution obtains the standard operation solution of a series of dicyandiamides, the concentration of dicyandiamide is respectively 5,10,25 in the described standard operation solution, 50,100ng/mL, the difference sample introduction, obtain chromatographic peak area, with chromatographic peak area the mass concentration of dicyandiamide is done linear regression, the typical curve that obtains dicyandiamide is: Y=17.5x+60.24, r=0.9994, the range of linearity is 5-100ng/mL, the good (see figure 2) of linear relationship.
3. the mensuration of sample
Embodiment 1
The described powdered milk sample of present embodiment is the whole milk powder negative sample, and described liquid-matter coupling detects the method for dicyandiamide in the milk powder, specifically comprises the steps:
The pre-treatment of a, sample, and specifically comprise the steps:
(1) takes by weighing the 1g powdered milk sample, add 2mL water and make the milk powder dissolving, add 10mL acetonitrile extract, under the ultrasound wave of 40kHz, extracted 10 minutes, obtain mixed solution;
(2) in described mixed solution, add 2gMgSO
4And 0.5gNaCl, the first centrifugal supernatant is carried out in vortex vibration 1 minute then on the hydro-extractor of 7000rpm;
(3) get the described first supernatant 5mL, transfer in the glass test tube that contains 150mg adsorbent C18 (particle diameter is 40-60 μ m), vortex 1min carries out centrifugal second supernatant that gets in 1500rpm then so that adsorbent and solution mix;
(4) get the described second supernatant 4mL, be transferred in the clean glass test tube, after drying up of 45 ℃ of nitrogen concentrates, mixed liquor with 0.80mL acetonitrile and 0.1% aqueous formic acid redissolves, the volume ratio of acetonitrile described in the described mixed liquor and 0.1% aqueous formic acid is 3:7, cross aperture 0.22 μ m filter membrane at last, make testing sample.
B, employing Ultra Performance Liquid Chromatography-triple level Four bar GC-MS detect the dicyandiamide that contains in the described testing sample:
(1) described high-efficient liquid phase chromatogram condition is:
Chromatographic column: Waters ACQUITY UPLC HSS T32.1 * 150mm, 1.8 μ m chromatographic columns;
Column temperature: 30 ℃;
Mobile phase A: 10mmol/LNH
4The Ac aqueous solution, Mobile phase B: acetonitrile;
The gradient elution program:
Flow velocity is 0.3mL/min;
Sampling volume: 10.0L.
(2) described mass spectrum condition is:
Mass analyzer: triple level Four bars;
Ion gun: ESI
+
Detecting pattern: multiple-reaction monitoring pattern (MRM);
Capillary voltage: 4kV;
Taper hole voltage: 25V;
Ion source temperature: 150 ℃;
Desolventizing temperature: 400 ℃;
Center gas flow velocity: 50L/Hr;
Desolventizing gas flow rate: 800L/Hr;
Collision gas velocity: 0.15mL/min.
Wherein, parameters such as monitoring ion pair, taper hole voltage and collision energy etc. are as shown in table 1.
The triple level Four bar of table 1 The MS detection parameters
Shown in Fig. 3 A, Fig. 3 B, be respectively dicyandiamide in the whole milk powder negative sample total ion current and the selection ion figure of daughter ion m/z68, dicyandiamide is not for detecting in this sample.
Embodiment 2
The described milk powder of present embodiment is the skimmed milk power positive, and described liquid-matter coupling detects the sample method of dicyandiamide in the milk powder, specifically comprises the steps:
The pre-treatment of a, sample, and specifically comprise the steps:
(1) takes by weighing the 1g powdered milk sample, add 2mL water and make the milk powder dissolving, add 10mL acetonitrile extract, under the ultrasound wave of 40kHz, extracted 10 minutes, obtain mixed solution;
(2) in described mixed solution, add 3gMgSO
4And 1gNaCl, the first centrifugal supernatant is carried out in vortex vibration 1 minute then on the hydro-extractor of 7000rpm;
(3) get the described first supernatant 5mL, transfer to that to contain 200mg adsorbent C18(particle diameter be 40-60 μ m) glass test tube in, vortex 1min carries out centrifugal second supernatant in 1500rpm then so that adsorbent and solution mix;
(4) get the described second supernatant 4mL, be transferred in the clean glass test tube, after drying up of 45 ℃ of nitrogen concentrates, mixed liquor with 0.80mL acetonitrile and 0.1% aqueous formic acid redissolves, the volume ratio of acetonitrile described in the described mixed liquor and 0.1% aqueous formic acid is 3:7, cross aperture 0.22 μ m filter membrane at last, make testing sample.
B, employing Ultra Performance Liquid Chromatography-triple level Four bar GC-MS detect the dicyandiamide that contains in the described testing sample:
(1) described high-efficient liquid phase chromatogram condition:
Chromatographic column: Waters ACQUITY UPLC HSS T32.1 * 150mm, 1.8 μ m chromatographic columns;
Column temperature: 30 ℃;
Mobile phase A: 10mmol/LNH
4The Ac aqueous solution, Mobile phase B: acetonitrile;
The gradient elution program:
Flow velocity is 0.3mL/min;
Sampling volume: 10.0L.
(2) described mass spectrum condition is:
Mass analyzer: triple level Four bars;
Ion gun: ESI
+
Detecting pattern: multiple-reaction monitoring pattern (MRM);
Capillary voltage: 4kV;
Taper hole voltage: 25V;
Ion source temperature: 150 ℃;
Desolventizing temperature: 400 ℃;
Center gas flow velocity: 50L/Hr;
Desolventizing gas flow rate: 800L/Hr;
Collision gas velocity: 0.15mL/min.
Wherein, parameter values such as monitoring ion pair, taper hole voltage and collision energy are identical with embodiment 1, as shown in table 1.
Shown in Fig. 4 A, Fig. 4 B, be respectively dicyandiamide in the whole milk powder positive total ion current and the selection ion figure of daughter ion m/z68, adopt external standard method with peak area quantification, the content that records dicyandiamide is 0.30mg/kg.
Embodiment 3
The described milk powder of present embodiment is the skimmed milk power positive, and described liquid-matter coupling detects the sample method of dicyandiamide in the milk powder, specifically comprises the steps:
The pre-treatment of a, sample, and specifically comprise the steps:
(1) takes by weighing the 1g powdered milk sample, add 2mL water and make the milk powder dissolving, add 10mL acetonitrile extract, under the ultrasound wave of 40kHz, extracted 10 minutes, obtain mixed solution;
(2) in described mixed solution, add 2gMgSO
4And 0.5gNaCl, the first centrifugal supernatant is carried out in vortex vibration 1 minute then on the hydro-extractor of 7000rpm;
(3) get the described first supernatant 5mL, transfer to that to contain 150mg adsorbent C18(particle diameter be 40-60 μ m) glass test tube in, vortex 1min carries out centrifugal second supernatant in 1500rpm then so that adsorbent and solution mix;
(4) get the described second supernatant 4mL, be transferred in the clean glass test tube, after drying up of 45 ℃ of nitrogen concentrates, mixed liquor with 0.80mL acetonitrile and 0.1% aqueous formic acid redissolves, the volume ratio of acetonitrile described in the described mixed liquor and 0.1% aqueous formic acid is 3:7, cross aperture 0.22 μ m filter membrane at last, make testing sample.
B, employing Ultra Performance Liquid Chromatography-triple level Four bar GC-MS detect the dicyandiamide that contains in the described testing sample:
(1) described high-efficient liquid phase chromatogram condition:
Chromatographic column: Waters ACQUITY UPLC HSS T32.1 * 150mm, 1.8 μ m chromatographic columns;
Column temperature: 30 ℃;
Mobile phase A: 10mmol/LNH
4The Ac aqueous solution, Mobile phase B: acetonitrile;
The gradient elution program:
Flow velocity is 0.3mL/min;
Sampling volume: 10.0L.
(2) described mass spectrum condition is:
Mass analyzer: triple level Four bars;
Ion gun: ESI
+
Detecting pattern: multiple-reaction monitoring pattern (MRM);
Capillary voltage: 4kV;
Taper hole voltage: 25V;
Ion source temperature: 150 ℃;
Desolventizing temperature: 400 ℃;
Center gas flow velocity: 50L/Hr;
Desolventizing gas flow rate: 800L/Hr;
Collision gas velocity: 0.15mL/min.
Wherein, parameter values such as monitoring ion pair, taper hole voltage and collision energy are identical with embodiment 1, as shown in table 1.
The total ion current and the daughter ion m/z68 that are respectively dicyandiamide in the skimmed milk power positive shown in Fig. 5 A, 5B select ion figure, adopt external standard method with peak area quantification, and the content that records dicyandiamide is 0.40mg/kg.
Embodiment 4
The described milk powder of present embodiment is the baby milk powder negative sample, and described liquid-matter coupling detects the method for dicyandiamide in the milk powder, specifically comprises the steps:
The pre-treatment of a, sample, and specifically comprise the steps:
(1) takes by weighing the 1g powdered milk sample, add 2mL water and make the milk powder dissolving, add 10mL acetonitrile extract, under the ultrasound wave of 40kHz, extracted 10 minutes, obtain mixed solution;
(2) in described mixed solution, add 1gMgSO
4And 0.5gNaCl, the first centrifugal supernatant is carried out in vortex vibration 1 minute then on the hydro-extractor of 7000rpm;
(3) get the described first supernatant 5mL, transfer in the glass test tube that contains 300mg adsorbent C18, vortex 1min carries out centrifugal second supernatant that gets in 1500rpm then so that adsorbent and solution mix;
(4) get the described second supernatant 4mL, be transferred in the clean glass test tube, after drying up of 45 ℃ of nitrogen concentrates, mixed liquor with 0.80mL acetonitrile and 0.1% aqueous formic acid redissolves, the volume ratio of acetonitrile described in the described mixed liquor and 0.1% aqueous formic acid is 3:7, cross aperture 0.22 μ m filter membrane at last, make testing sample.
B, employing Ultra Performance Liquid Chromatography-triple level Four bar GC-MS detect the dicyandiamide that contains in the described testing sample:
(1) described high-efficient liquid phase chromatogram condition:
Chromatographic column: Waters ACQUITY UPLC HSS T32.1 * 150mm, 1.8 μ m chromatographic columns;
Column temperature: 30 ℃;
Mobile phase A: 10mmol/LNH
4The Ac aqueous solution, Mobile phase B: acetonitrile;
The gradient elution program:
Flow velocity is 0.3mL/min;
Sampling volume: 10.0L.
(2) described mass spectrum condition:
Mass analyzer: triple level Four bars;
Ion gun: ESI
+
Detecting pattern: multiple-reaction monitoring pattern (MRM);
Capillary voltage: 4kV;
Taper hole voltage: 25V;
Ion source temperature: 150 ℃;
Desolventizing temperature: 400 ℃;
Center gas flow velocity: 50L/Hr;
Desolventizing gas flow rate: 800L/Hr;
Collision gas velocity: 0.15mL/min.
Wherein, parameter values such as monitoring ion pair, taper hole voltage and collision energy are identical with embodiment 1, as shown in table 1.
The total ion current and the daughter ion m/z68 that are respectively dicyandiamide in the baby milk powder negative sample shown in Fig. 6 A, 6B select ion figure, adopt external standard method with peak area quantification, and dicyandiamide is not for detecting in this sample.
4. the checking of method:
4.1 minimum detectability and minimum quantitative limit
The standard solution of a certain amount of dicyandiamide is joined in the powdered milk sample, make the concentration of adding dicyandiamide in the milk powder of back be respectively 5,10,25,50,100ng/g, the sample-pretreating method and the detection method that detect dicyandiamide in the milk powder according to the embodiment of the invention 1 described liquid-matter coupling experimentize.
According to signal to noise ratio (S/N ratio) S/N 〉=3 is the minimum detectability of this material, and detecting of dicyandiamide is limited to 10ng/g in the milk powder, is the minimum quantitative limit of this material according to signal to noise ratio (S/N ratio) S/N 〉=10, the minimum 30ng/g that quantitatively is limited to.
4.2 degree of accuracy
Successively accurately take by weighing the powdered milk sample (being accurate to 0.01g) of three parts of 1g, and number A-1, A-2, A-3 respectively, according to the embodiment of the invention 1 described method sample is carried out pre-treatment and detection, described A-1, A-2, A-3 powdered milk sample are carried out replicate determination, analysis result sees Table 2, and the relative standard deviation of dicyandiamide is 5.67% in the parallel sample; Further, first sample A-1 is carried out replication 5 times, analysis result sees Table 3, and the relative standard deviation of reperformance test is 3.05%.
The replicate determination of table 2 sample
The replication of table 3A-1 sample
4.3 the recovery
The standard solution of dicyandiamide is joined in the blank powdered milk sample (being the negative sample of milk powder), control mark-on concentration is respectively 25,50,100ng/g, the sample-pretreating method and the detection method that detect dicyandiamide in the milk powder according to the embodiment of the invention 1 described liquid-matter coupling experimentize, carry out 6 horizontal surveies under each mark-on concentration, average, according to the actual scalar sum testing result that adds, calculate the recovery of standard addition of powdered milk sample, see Table 4, the recovery of standard addition of sample is 95.2%～105.7%.
The horizontal survey of table 4 mark-on sample
Comparative Examples 1
The described powdered milk sample of this Comparative Examples is that 100ng/g makes for baby milk powder negative sample among the embodiment 4 adds the dicyandiamide amount, adopt with embodiment 4 identical Ultra Performance Liquid Chromatographies-triple level Four bar GC-MS the dicyandiamide that contains in the described testing sample is detected, difference only is sample is carried out not adding MgSO in described mixed solution in the step (2) of pre-treatment
4And NaCl, but the direct vortex of the mixed liquor after will extracting vibration 1 minute, the first centrifugal supernatant carries out on the hydro-extractor of 7000rpm in the back.
Under the interpolation level of 100ng/g, the content that records dicyandiamide in the powdered milk sample is 59.7ng/g, and promptly adding the recovery is 59.7%, therefore, adopts above-mentioned sample-pretreating method, will cause the recovery on the low side, and measuring error is bigger.
Comparative Examples 2
The described powdered milk sample of this Comparative Examples is that 100ng/g makes for baby milk powder negative sample among the embodiment 4 adds the dicyandiamide amount, adopt with embodiment 4 identical Ultra Performance Liquid Chromatographies-triple level Four bar GC-MS the dicyandiamide that contains in the described testing sample is detected, difference only is that the step (2) of sample being carried out pre-treatment is: add 0.5gMgSO in described mixed solution
4And 2gNaCl, the first centrifugal supernatant is carried out in vortex vibration 1 minute then on the hydro-extractor of 7000rpm.
Under the interpolation level of 100ng/g, the content that records dicyandiamide in the powdered milk sample is 61.8ng/g, and promptly adding the recovery is 61.8%, therefore, adopts above-mentioned sample-pretreating method, will cause the recovery on the low side, and measuring error is bigger.
Comparative Examples 3
The described powdered milk sample of this Comparative Examples is that 100ng/g makes for baby milk powder negative sample among the embodiment 4 adds the dicyandiamide amount, adopt with embodiment 4 identical Ultra Performance Liquid Chromatographies-triple level Four bar GC-MS the dicyandiamide that contains in the described testing sample is detected, difference only is the pre-treating method difference of sample.
The sample-pretreating method that this Comparative Examples adopts, its concrete steps are:
Take by weighing the 1g powdered milk sample, add 2mL water and make the milk powder dissolving, add 75mgEDTA-Na
2Mixing, add 2.5mL acetonitrile-alcohol mixeding liquid (90:10 again, v/v) mixing 30s, 0 ℃ of refrigerated centrifuge 10min obtains first supernatant, get 2.25mL first supernatant in centrifuge tube, add 15.5mL acetonitrile-alcohol mixeding liquid, abundant mixing, 0 ℃ of refrigerated centrifuge obtains second supernatant, second supernatant is transferred to 50mL heart bottle, 40 ℃ of rotary evaporations are settled to 0.5mL with acetonitrile-alcohol-water mixed liquor and obtain sample liquid to doing, and shake up back sample liquid and obtain liquid to be measured by 0.22 μ m filtering with microporous membrane.
Under the interpolation level of 100ng/g, the content that records dicyandiamide in the powdered milk sample is 56ng/g, and promptly adding the recovery is 56%, therefore, adopts above-mentioned sample-pretreating method, will cause the recovery on the low side, and measuring error is bigger.
Comparative Examples 4
The described powdered milk sample of this Comparative Examples is that 100ng/g makes for baby milk powder negative sample among the embodiment 4 adds the dicyandiamide amount, adopt with embodiment 4 identical Ultra Performance Liquid Chromatographies-triple level Four bar GC-MS the dicyandiamide that contains in the described testing sample is detected, difference only is the pre-treating method difference of sample.
The sample-pretreating method that this Comparative Examples adopts, its concrete steps are:
(1) takes by weighing the 1g powdered milk sample to the centrifuge tube of 50mL; (2) volume ratio of adding 25mL is the water of 1:9 and the mixed liquor of acetonitrile; (3) eddy current concussion 2min, ultrasonic 30min shakes frequently; (4) take out, through the centrifugal 4min of 10000r/min; (5) centrifuged supernatant obtains liquid to be measured through 0.22 μ m filtering with microporous membrane.
Under the interpolation level of 100ng/g, the content that records dicyandiamide in the powdered milk sample is 189.0ng/g, and promptly adding the recovery is 189%, therefore, adopts above-mentioned sample-pretreating method, will cause the recovery higher, and it is inaccurate to record the result.
Sample-pretreating method of the present invention, with organic extract sample is carried out the extraction of dicyandiamide after, with MgSO
4With NaCl sample being carried out layering extracts, utilize the increase of salinity in the water, further reduce dicyandiamide, and then increase its concentration of ordinary dissolution in organic phase in aqueous phase solubleness, it is distributed by liquid liquid and more enter organic phase, the extraction efficiency of maximized increase dicyandiamide; Simultaneously because MgSO
4Solubleness in water is less, and has very strong water absorptivity, and produces spontaneous heating in suction, can further increase the solubleness of dicyandiamide in organic phase, thereby improves extraction efficiency; In addition, by means of MgSO
4With the salting out of NaCl, promote organic phase and water layering, improve separation efficiency; And by to the absorption, separation of extracting back solution, concentrate, the processing of steps such as redissolution, filtration, farthest may disturb the factor that detects the result to handle, further promoted detection accuracy and detection sensitivity.
Preferably, when the quality that takes by weighing powdered milk sample is 1g, described MgSO
4Consumption be 1-3g, and preferred 2g had both avoided MgSO
4Consumption causes suction and exothermic effect more weak very little, does not have the problem that improves extraction efficiency, has also avoided MgSO
4Consumption is too big, will produce the caking of bulky grain shape, causes MgSO
4Disperse inhomogeneously with sample substrate, influence the problem of extraction efficiency; And studies show that, at preferred MgSO
4Under the condition of consumption, the MgSO of dispersion
4Solid particle and milk powder matrix form mixture, and play friction and exothermic effects in the vortex process, have improved extraction efficiency effectively; The consumption of described NaCl is 0.5-1g, and preferred 0.5g, both solved the NaCl consumption very little, cause salting out not obvious, and consumption is excessive, have part NaCl and enter acetonitrile layer, follow-up dry up have in the process part salt out, the excessive concentration of salt can suppress the problem of mass spectrum response signal, and efficient but also guaranteed follow-up detection accuracy has not only guaranteed to saltout.
Obviously, the foregoing description only is for example clearly is described, and is not the qualification to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here need not also can't give exhaustive to all embodiments.And conspicuous variation of being extended out thus or change still are among the protection domain of the invention.
Claims (12)
1. a liquid-matter coupling detects the sample-pretreating method of dicyandiamide in the milk powder, it is characterized in that, comprises the following steps:
(1) powdered milk sample is dissolved aqueous solution, and add organic extract dicyandiamide extracted;
(2) add MgSO in the mixed liquor after extracting
4Make described organic extract and water layering with NaCl, the centrifugal supernatant of winning;
(3) in described first supernatant, add adsorbent and purify, centrifugal second supernatant that gets;
(4) with described second supernatant through concentrate, redissolution, filtration step, obtain required testing sample.
2. liquid according to claim 1-matter coupling detects the sample-pretreating method of dicyandiamide in the milk powder, it is characterized in that, and in the described step (2), described MgSO
4Addition and the mass ratio of described powdered milk sample be 1:1-3:1.
3. liquid according to claim 2-matter coupling detects the sample-pretreating method of dicyandiamide in the milk powder, it is characterized in that the addition of described NaCl and the mass ratio of described powdered milk sample are 0.5:1-1:1.
4. detect the sample-pretreating method of dicyandiamide in the milk powder according to the arbitrary described liquid of claim 1-3-matter coupling, it is characterized in that, in the described step (1), the step that described organic extract extracts dicyandiamide is carried out under ultrasound condition.
5. detect the sample-pretreating method of dicyandiamide in the milk powder according to the arbitrary described liquid of claim 1-4-matter coupling, it is characterized in that, in the described step (1), described organic extract is one or more the potpourri in acetonitrile, acidifying acetonitrile, ammonification acetonitrile, ethyl acetate, methyl alcohol or the ethanol.
6. detect the sample-pretreating method of dicyandiamide in the milk powder according to the arbitrary described liquid of claim 1-5-matter coupling, it is characterized in that in the described step (3), described adsorbent is the C18 adsorbent of particle diameter 40-60 μ m.
7. liquid according to claim 6-matter coupling detects the sample-pretreating method of dicyandiamide in the milk powder, it is characterized in that the addition of described adsorbent and the mass ratio of described powdered milk sample are 0.15:1-0.3:1.
8. detect the sample-pretreating method of dicyandiamide in the milk powder according to the arbitrary described liquid of claim 1-7-matter coupling, it is characterized in that, in the described step (4), described concentration step adopts the mode that dries up with nitrogen to carry out.
9. liquid according to claim 8-matter coupling detects the sample-pretreating method of dicyandiamide in the milk powder, it is characterized in that, described redissolution step is to adopt the mixed liquor of the aqueous formic acid of acetonitrile and 0.1% to carry out, and the volume ratio of described acetonitrile and described formic acid solution is 3:7.
10. a liquid-matter coupling detects the method for dicyandiamide in the milk powder, it is characterized in that, comprises the steps:
(a) according to the arbitrary described method of claim 1-9 described milk powder is carried out pre-treatment, obtain required testing sample;
(b) adopt Ultra Performance Liquid Chromatography-triple level Four bar GC-MS that the dicyandiamide that contains in the milk powder is detected.
11. liquid according to claim 10-matter coupling detects the method for dicyandiamide in the milk powder, it is characterized in that the testing conditions of high performance liquid chromatography is described in the described step (b):
Chromatographic column: Waters ACQUITY UPLC HSS T32.1 * 150mm, 1.8 μ m;
Column temperature: 30 ℃;
Mobile phase A: 10mmol/LNH
4The Ac aqueous solution, Mobile phase B: acetonitrile;
Flow velocity: 0.25mL/min;
Sample size: 10 μ L;
Type of elution: gradient elution;
The mass spectral testing conditions of described triple level Four bars is:
Mass analyzer: triple level Four bars;
Ion gun: ESI
+
Capillary voltage: 4kV;
Taper hole voltage: 25V;
Ion source temperature: 150 ℃;
Desolventizing temperature: 400 ℃;
Center gas flow velocity: 50L/Hr;
Desolventizing gas flow rate: 800L/Hr;
Collision gas velocity: 0.15mL/min.
12. liquid according to claim 11-matter coupling detects the method for dicyandiamide in the milk powder, it is characterized in that the gradient elution program of described high performance liquid chromatography is:
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107271576A (en) * | 2016-04-08 | 2017-10-20 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method for determining dicyandiamide residual quantity in dairy products |
| CN109187832A (en) * | 2018-09-30 | 2019-01-11 | 华润三九医药股份有限公司 | LC-MS/MS measures the method for neo-synephrine concentration and the pre-treating method of sample in blood plasma |
| CN111521458A (en) * | 2020-04-21 | 2020-08-11 | 深圳市深大检测有限公司 | Method for detecting liquid milk melamine |
| CN115078607A (en) * | 2022-06-21 | 2022-09-20 | 浙江省农业科学院 | Sample pretreatment method for determining perfluorinated compounds in dairy products based on magnetic nano material one-step method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102323347A (en) * | 2011-07-28 | 2012-01-18 | 福建省农业科学院土壤肥料研究所 | Method for measuring dicyandiamide in soil and water body |
| CN102419354A (en) * | 2011-09-08 | 2012-04-18 | 宁波检验检疫科学技术研究院 | General rapid detection method for micromolecular poisonous and harmful substances in liquid milk |
-
2013
- 2013-04-23 CN CN201310142797.4A patent/CN103217498B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102323347A (en) * | 2011-07-28 | 2012-01-18 | 福建省农业科学院土壤肥料研究所 | Method for measuring dicyandiamide in soil and water body |
| CN102419354A (en) * | 2011-09-08 | 2012-04-18 | 宁波检验检疫科学技术研究院 | General rapid detection method for micromolecular poisonous and harmful substances in liquid milk |
Non-Patent Citations (5)
| Title |
|---|
| SHAUN MACMAHON ET AL.: "A liquid chromatography–tandem mass spectrometry method for the detection of economically motivated adulteration in protein-containing foods", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| 丁娟娟 等: "沃特世针对乳制品中双氰胺测定的整体解决方案", 《食品安全导刊》 * |
| 吴翠玲 等: "安捷伦QuEChERS前处理方法结合LC-MS/MS快速检测乳品中双氰胺", 《食品安全导刊》 * |
| 郑军红: "LC-MS/MS测定牛奶和奶粉中多种农药残留量的研究", 《中国优秀硕士学位论文全文数据库》 * |
| 黄芳 等: "液相色谱-串联质谱法快速测定奶粉中的双氰胺", 《分析测试学报》 * |
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|---|---|---|---|---|
| CN107271576A (en) * | 2016-04-08 | 2017-10-20 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method for determining dicyandiamide residual quantity in dairy products |
| CN109187832A (en) * | 2018-09-30 | 2019-01-11 | 华润三九医药股份有限公司 | LC-MS/MS measures the method for neo-synephrine concentration and the pre-treating method of sample in blood plasma |
| CN109187832B (en) * | 2018-09-30 | 2021-07-30 | 华润三九医药股份有限公司 | Method for determining phenylephrine concentration by LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) and sample pretreatment method |
| CN111521458A (en) * | 2020-04-21 | 2020-08-11 | 深圳市深大检测有限公司 | Method for detecting liquid milk melamine |
| CN111521458B (en) * | 2020-04-21 | 2023-02-03 | 深圳市深大检测有限公司 | Method for detecting liquid milk melamine |
| CN115078607A (en) * | 2022-06-21 | 2022-09-20 | 浙江省农业科学院 | Sample pretreatment method for determining perfluorinated compounds in dairy products based on magnetic nano material one-step method |
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