CN101776664A - Method for testing melamine in raw milk and dairy products and preprocessing device - Google Patents
Method for testing melamine in raw milk and dairy products and preprocessing device Download PDFInfo
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- CN101776664A CN101776664A CN201010109986A CN201010109986A CN101776664A CN 101776664 A CN101776664 A CN 101776664A CN 201010109986 A CN201010109986 A CN 201010109986A CN 201010109986 A CN201010109986 A CN 201010109986A CN 101776664 A CN101776664 A CN 101776664A
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- 238000000034 method Methods 0.000 title claims abstract description 62
- 229920000877 Melamine resin Polymers 0.000 title claims abstract description 50
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 235000013365 dairy product Nutrition 0.000 title claims abstract description 24
- 235000020185 raw untreated milk Nutrition 0.000 title claims abstract description 20
- 238000012360 testing method Methods 0.000 title claims abstract description 20
- 238000007781 pre-processing Methods 0.000 title abstract 2
- 239000000523 sample Substances 0.000 claims abstract description 57
- 239000012528 membrane Substances 0.000 claims abstract description 46
- 239000000835 fiber Substances 0.000 claims abstract description 38
- 239000012071 phase Substances 0.000 claims abstract description 32
- 239000012074 organic phase Substances 0.000 claims abstract description 27
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 26
- 238000001334 liquid-phase micro-extraction Methods 0.000 claims abstract description 24
- 239000012488 sample solution Substances 0.000 claims abstract description 21
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 239000012510 hollow fiber Substances 0.000 claims abstract description 8
- 238000000605 extraction Methods 0.000 claims description 30
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical group CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 claims description 26
- 235000013336 milk Nutrition 0.000 claims description 26
- 239000008267 milk Substances 0.000 claims description 26
- 210000004080 milk Anatomy 0.000 claims description 26
- 238000001514 detection method Methods 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 17
- 238000002203 pretreatment Methods 0.000 claims description 17
- 239000000843 powder Substances 0.000 claims description 12
- 238000007789 sealing Methods 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
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- 239000004698 Polyethylene Substances 0.000 claims description 2
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- 238000004587 chromatography analysis Methods 0.000 abstract description 2
- 239000011148 porous material Substances 0.000 abstract 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 12
- 235000013305 food Nutrition 0.000 description 10
- 239000003795 chemical substances by application Substances 0.000 description 8
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- 238000010561 standard procedure Methods 0.000 description 8
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
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- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 description 4
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- 241000282326 Felis catus Species 0.000 description 1
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Abstract
The invention discloses a method for testing melamine in raw milk and dairy products. The high-efficient liquid phase chromatography is adopted for testing, the samples of raw milk and dairy product are preprocessed by way of hollow fiber membrane liquid-liquid-liquid phase microextraction, the organic phase is adsorbed on the pore wall of the fiber membrane, the aqueous phase as the receiving phase exists in the fiber membrane, and after being soaked in the sample solution to be detected and is extracted, the fiber membrane is directly tested by HPLC. Meanwhile, the invention also discloses a sample preprocessing device for the method and a preparation method thereof. The method is sensitive and reliable, the operation of the testing method is simple and convenient, the cost is saved, and the method is environment-friendly.
Description
Technical field
The invention belongs to the food inspection field, relate to a kind of liquid-phase microextraction method that adopts and carry out the method that organism is analyzed, more specifically say so and adopt hollow-fibre membrane three-phase liquid-phase micro-extraction-high performance liquid chromatography to carry out the mensuration of melamine in raw milk and the dairy products.
Background technology
Melamine is a kind of important chemical material originally, is the common used in industry chemicals.Be separated by very far away with field of food.So the contaminated incident of Sanlu baby milk powder still occurring in China in September, 2008 but makes one time of melamine become the milk accessory.Melamine detection in milk or the dairy produce, the major issue that has become domestic and international analytical chemistry field to pay close attention to suddenly, closely bound up with food safety detection.
Because melamine originally is an industrial chemicals, does not allow to add in the food, so before this, the national standard of melamine in the food does not all detect in various countries, and detection method is comparatively backward.For melamine detection, traditional have nephelometry, sublimed method, a gravimetric method, and these methods are mainly the mensuration amine of chemical industry, and it is used to measure the purity of synthetic melamine, can not satisfy the requirement that detects micro-melamine in the food; Whether Enzyme Linked Immunoadsorbent Assay (ELISA) is mainly by containing melamine in kit or the direct working sample of test paper, can satisfy the requirement that detects melamine in the law enforcement of daily bread health, but this method is subjected to the interference of other materials easily, influence the accuracy of testing result, the general initial survey that only is applicable to sample need adopt the chemical apparatuses method further to prove conclusively for the sample of positive findings.
High performance liquid chromatography (HPLC), high performance liquid chromatography-mass spectrometry method (LC-MS), vapor-phase chromatography-mass spectrometry method (GC-MS) are high-precision test methods in the analyzing and testing field in recent years, the melamine that it is used for detecting food has the advantage highly sensitive, that accuracy is strong.In the U.S., FDA has announced the detailed process that detects melamine with GC-MS and HPLC in succession after the melamine in March, 2007 pollutes cat grain dog grain incident.But the detection of adopting liquid chromatography, gas chromatography etc. to carry out need be carried out numerous and diverse pre-treatment process to food samples.With China national standard method GB/T 22388-2008 (HPLC method) (hereinafter to be referred as " National Standard Method ") is example, it comprises following pre-treatment process: solution extraction-centrifuging-filter paper filtering-SPE column purification-eluent concentrates, constant volume-filtering with microporous membrane, just can enter HPLC at last and analyze.These or detection method loaded down with trivial details or that reliability is low can not adapt to melamine detection pressure in the present field of food.How in the advantage of utilizing liquid phase chromatography, vapor-phase chromatography high precision and high sensitivity, overcome the deficiency that takes time and effort that its pre-treatment brings, become the important problem of this detection range.
Summary of the invention
The objective of the invention is to be at the deficiencies in the prior art, the method for melamine in a kind of sensitive and accurate and fast and convenient detection raw milk and the dairy products is provided.
Another object of the present invention is to provide the sample pre-treatments device of this method.
The present invention is achieved through the following technical solutions above-mentioned purpose:
The invention provides a kind of method that detects melamine in raw milk and the dairy products, adopt high performance liquid chromatography to measure, before entering high performance liquid chromatography, sample carries out pre-treatment by adopting hollow-fibre membrane three-phase liquid-phase micro-extraction.
(Hollow fiber liquid phase microextraction HF-LPME) is liquid-phase micro-extraction technology (Liquid phase microextraction, a kind of in LPME) to the hollow-fibre membrane liquid-phase micro-extraction.The liquid-phase micro-extraction technology is a kind of novel sample-pretreating method, also can be easily combines with follow-up instrumental analysis, realizes the integrated process of sample pre-treatments and assay determination.Difference by the extraction mode can be divided into headspace liquid-phase microextraction (HS-LPME), directly immersion liquid-phase micro-extraction (DI-LPME) and hollow-fibre membrane liquid-phase micro-extraction (HF-LPME).Wherein, the headspace liquid-phase microextraction composition that requires to be extracted must be volatile; Directly the immersion liquid-phase micro-extraction then is applicable to comparatively clean liquid phase sample; And the hollow-fibre membrane liquid-phase micro-extraction is for complicated component, and the more or coloured liquid phase sample of impurity content is particularly suitable.
Although LPME is applied to various materials gradually, in the pre-treating method as environment, biological sample, food etc.But, any relevant report that LPME is applied to the melamine detection is not arranged at present yet.Before the present invention, adopt the feasibility of LPME, and how to implement, as yet unknown number not as the pre-treatment means that detect melamine.
The present invention creatively with the HF-LPME among the LPME as the pre-treatment means that detect melamine in raw milk and the dairy products, and find out a cover and can efficiently extract melamine in the sample solution, and be suitable for directly entering pre-treating method and the system that HPLC detects.
HF-LPME be at first 1999 by propositions such as Pedersen-Bjergaard, be to carry out mass transfer by the liquid film that organic solvent forms in the fiber cinclides, in the hollow fiber chamber of porous, extract.HF-LPME has two-phase extraction and two kinds of patterns of three phase extraction.In the two-phase extraction pattern, measured object is by being fixed on water-fast organic phase in the hollow fiber membrane micropore, enter in the film chamber reception mutually.This moment, organic phase was identical organic solvent with receiving mutually, and the enrichment process of test substance occurs between the two-phase.And in the three phase extraction pattern, reception in the hollow-fibre membrane chamber is not organic phase but water mutually, and test substance enters into aqueous phase in the film then by being fixed on the organic phase in the fenestra.
The present invention finds, organic phase is fixed in the fenestra,, is particularly suitable for extracting the melamine in the sample solution and directly enters the HPLC detection system as receiving phase with water.Adopt hollow-fibre membrane three-phase micro-extraction technique to make object must be introduced into the organic phase of membranous wall, enter into the interior reception of film mutually, have more outstanding purification function and antijamming capability than two-phase extraction.
Detection method provided by the invention is as follows:
Raw milk and dairy products preparation become after the solution, with hollow-fibre membrane three-phase liquid-phase micro-extraction the melamine in the solution are extracted, and directly enter HPLC again and detect.In this method, the three-phase liquid-phase micro-extraction is in hollow-fibre membrane, as receiving the inside that is present in tunica fibrosa mutually, is filled with organic phase on the hole wall of tunica fibrosa with water; Hollow-fibre membrane is positioned in the testing sample solution, promptly carries out extraction process.
In the method, tunica fibrosa is Kynoar tunica fibrosa, polyethylene fibre film or polypropylene screen tunica fibrosa; It is the thin tube-like of hollow, and two ends can be sealed, its wall thickness 100～300 μ m, internal diameter 1.2～10mm, aperture 0.1～0.3 μ m; Organic phase is full of the hole wall of tunica fibrosa by certain processing; Organic phase is a water-insoluble organic solvent, as n-octyl alcohol, toluene.
Hollow-fibre membrane is positioned over the time that extracts in the testing sample solution and can be 30～120min; Extraction process is to carry out under the rotating speed of 500～1200rpm.
Adopt the method, can detect plurality of raw materials breast and dairy products, as liquid milk, milk powder.Powder or solid sample must detect after being treated as solution through certain again.Such as, powdered milk sample makes sample solution by adding water ultrasonic dissolution method.Other dairy products such as cheese take by weighing sample and grind to form the dry powder shape in mortars, become sample solution behind the water ultrasonic dissolution and detect by adding.
The present invention provides the sample pre-treatments that is applicable to this method device simultaneously, comprise the bottle with cover that to place testing sample solution, the lid below is hung with the tunica fibrosa coating that contains organic phase and water, and organic phase wherein is adsorbed in the hole wall of tunica fibrosa; Described coating at least a portion is soaked in the sample solution.
The present invention also provides the method for preparing said apparatus, may further comprise the steps:
(1) will clutch sealing by the end that hollow-fibre membrane constitutes, the other end links to each other with the plastics suction nozzle, becomes extracting tube, and the hollow-fibre membrane end is the extracting tube bottom, and plastics suction nozzle end is the extracting tube top;
(2) extracting tube is immersed in the organic phase solution, take out ultrasonic back, with lens wiping paper unnecessary organic phase is blotted;
(3) top of the extracting tube of step (2) gained is fixed in the bottle cap of sample bottle, when covering bottle cap, at least a portion of tunica fibrosa will be soaked in the sample solution in the sample bottle; And the interface of tunica fibrosa and suction nozzle is positioned on the sample solution.
(4) the interior water that injects of past pipe from the extracting tube top is full of it or part is full of the hollow fiber membrane portions, seals the extracting tube top again.
Concrete preparation process is as follows:
(1) prepare a bottle that has bottle cap and pad, according to the sample bottle height, the hollow-fibre membrane of cutting suitable length and transparent pipettor suction nozzle (specification is 0.5 μ L～10 μ L), the pipettor suction nozzle keeps tip portion, cuts off thick bore end., take out and dry the back and with glue hollow-fibre membrane and suction nozzle tip are connected and fixed to remove its dirt with acetone ultrasonic cleaning 20 minutes, the hollow-fibre membrane other end is clutched sealing with pliers;
(2) extracting tube is immersed in the organic phase ultrasonic 10 seconds, takes out the back and sops up organic phase unnecessary on the hollow-fibre membrane with lens wiping paper;
(3) by the pad in the bottle cap, extracting tube is inserted in the sample bottle, when covering bottle cap, at least a portion of tunica fibrosa will be soaked in the sample solution in the sample bottle; And the interface of tunica fibrosa and suction nozzle is positioned on the sample solution.
(4) drawing 20 μ L with microsyringe receives in phase (distilled water) the injection hollow-fibre membrane, with sealing the membrane closure mouth of pipe;
The above pretreating device that provides is positioned in the oscillator when extracting, and at room temperature with the rotating speed of 600r/min, extracts 90min; After extraction finishes, in film, draw all reception phases, be transferred in the sample introduction bottle, treat that HPLC surveys with microsyringe.
Adopt this method to carry out melamine detection in raw milk and the dairy products, not only easy and simple to handle, and saved experimental cost.Below be example with national standard method GB/T 22388-2008 (HPLC method), from operation steps (as table 1) with detect cost (as table 2) two aspects and compare.
The contrast of table 1. operation steps
| National standard method GB/T 22388-2008 | Method of the present invention |
| Solvent extraction | Make micro-extraction device |
| Centrifuging | The hollow-fibre membrane liquid-phase extraction |
| Filter paper filtering | HPLC analyzes |
| The SPE column purification |
| National standard method GB/T 22388-2008 | Method of the present invention |
| Eluent concentrates, constant volume | |
| Filtering with microporous membrane | |
| HPLC analyzes |
Table 2. detects the comparison of cost
By more as can be known, experimental implementation step of the present invention is few, only needs two kinds of instrument and equipments, and experimental cost is about 1/10 of National Standard Method, compares with National Standard Method, and advantage is outstanding.
Compared with prior art, the present invention has following beneficial effect:
The method of melamine is sensitive reliable in mensuration raw milk provided by the present invention and the dairy products, is carrier with the hollow fiber, has more increased the stability of extraction, has increased the surface area that solvent contacts with sample, has improved extraction efficiency, and is highly sensitive.
Assay method provided by the present invention is easy and simple to handle, carries out sample pre-treatments by micro-extraction technique, and centralized procurement sample and sample purification have been saved manpower and materials greatly in one.
Method provided by the present invention has significantly reduced testing process to the influence of environment and the waste of solvent, saves cost and environmentally friendly.By the mode of micro-extraction, method amount of samples of the present invention is few, environmentally friendly, and hollow fiber can only disposablely use, and has avoided the phenomenon of cross pollution.
Description of drawings
Fig. 1 sample pre-treatments device of the present invention: 1 for rubber sheet gasket, 2 for bottle cap, 3 for the extraction bottle, 4 for the plastics suction nozzle, 5 for hole wall be adsorbed with organic phase hollow-fibre membrane, 6 for sample solution, 7 for receiving phase.
The melamine standard colors spectrogram of Fig. 2 embodiment 2.
The liquid milk blank sample chromatogram of Fig. 3 embodiment 2.
The liquid milk mark-on sample chromatogram figure of Fig. 4 embodiment 2 (the interpolation level is 1 μ g/mL).
The milk powder blank sample chromatogram of Fig. 5 embodiment 3.
The milk powder mark-on sample chromatogram figure of Fig. 6 embodiment 3 (the interpolation level is 2 μ g/mL).
The extraction results of the sample pretreatment process of Fig. 7 embodiment 4: (A) be sample emulsion; (B) be the reception phase after the extraction.
The liquid milk blank sample chromatogram of Fig. 8 embodiment 5.
The liquid milk mark-on sample chromatogram figure of Fig. 9 embodiment 5 (the interpolation level is 0.05 μ g/mL).
Embodiment
Further specify content of the present invention by the following examples.
The preparation of sample pre-treatments device:
With hollow-fibre membrane be cut into 2.8 centimeter length the section, the material of hollow-fibre membrane is the Kynoar tunica fibrosa, wall thickness 200 μ m, internal diameter 1.2mm, aperture 0.2 μ m; The pipettor suction nozzle of getting 5～10 μ L is as the plastics suction nozzle, and suction nozzle cuts off thick mouthful of end, keeps the tip end, is cut into the section of 2.7 centimeter length.The film that shears and suction nozzle 4 are immersed in the acetone ultrasonic 20 minutes respectively, and taking-up is dried; Then that hollow-fibre membrane and suction nozzle 4 tips is glutinous tight with glue, the other end of clutching the sealing hollow-fibre membrane with blunt-ended forceps transparent impression occurs to film, promptly make extracting tube;
Extracting tube is immersed in the n-octyl alcohol (organic phase), take out after ultrasonic 10 seconds, make n-octyl alcohol be filled in the hollow-fibre membrane hole wall; Wipe n-octyl alcohol unnecessary on the extracting tube with lens wiping paper, this moment, the hypomere of extracting tube was the hollow-fibre membrane 5 that hole wall is adsorbed with organic phase.
Extracting tube is inserted the extraction bottle, and suction nozzle 4 other ends are by the pad 1 of bottle cap 2, and the position of fixed extractor pipe makes extracting tube be suspended in extraction bottle centre.
Add sample solution toward the extraction bottle, the hollow-fibre membrane 5 that hole wall is adsorbed with organic phase immerses sample liquid, and guarantees that hole wall is adsorbed with the hollow-fibre membrane 5 of organic phase and the interface of suction nozzle is higher than sample liquid, does not immerse in the sample.
Draw distilled water (reception phase) with micro syringe, inject the extracting tube inner chamber; Then with the mouth of pipe that seals membrane closure extracting tube suction nozzle 4.
The blank test of detection method and mark-on recovery test:
Preparation 1mg/mL melamine standard solution.Accurately measure 8mL liquid milk sample, the mark-on group adds 8 μ L melamine standard solution (i.e. interpolation level is 1 μ g/mL), and blank group does not add three samples of every group of parallel processing.Extracting tube was immersed in the n-octyl alcohol ultrasonic 10 seconds, takes out the back and sops up unnecessary n-octyl alcohol with lens wiping paper.Extracting tube is inserted in the sample bottle, draw 20 μ L distilled water (reception phase) with microsyringe and inject in the film, with sealing the membrane closure mouth of pipe.Extraction equipment is put into oscillator, and the speed oscillation with 600r/min under the room temperature extracts 90min.The extraction back is drawn and is all received phase, is transferred in the sample introduction bottle, treats that HPLC surveys.Take by weighing 1.01g sodium heptanesulfonate and 1.05g citric acid, be dissolved in water and be settled to 500mL, make ion-pairing agent.
The HPLC model is Tianjin, island LC-20A, and detecting device is diode array detector SPD-M20A.Chromatographic column is DiamonsilTM (two generations of a diamond) C18 chromatographic column, column length 250mm, internal diameter 4.6mm, particle diameter 5 μ m.The moving phase condition be acetonitrile/ion-pairing agent (10/90, V/V), flow velocity 1.2mL/min, 30 ℃ of column temperatures detect wavelength 240nm.With its response peak area and retention time mapping on chromatogram.
Experimental result such as table 3 and Fig. 2, Fig. 3, shown in Figure 4:
Blank and the mark-on measuring result of table 3 liquid milk matrix
Annotate: RSD is the relative standard deviation of peak area.
By the result as can be known: under this chromatographic condition, the noiseless thing of liquid milk matrix influences the qualitative and quantitative of melamine, and average recovery of standard addition is 109.9%, and relative standard deviation is 2.3%.Illustrate that this method can be used for the mensuration of melamine in fluid milk.
Detect powdered milk sample:
Preparation 1mg/mL melamine standard solution.One parcel (25g) commercially available dried milk powder is dissolved in the 200mL warm water, and ultrasonic 5min promptly is mixed with milk sample.Accurately measure the 8mL milk sample, the mark-on group adds 16 μ L melamine standard solution (i.e. interpolation level is 2 μ g/mL), and blank group does not add six samples of every group of parallel processing.Extracting tube was immersed in the n-octyl alcohol ultrasonic 10 seconds, takes out the back and sops up unnecessary n-octyl alcohol with lens wiping paper.Extracting tube is inserted in the sample bottle, draw 20 μ L distilled water (reception phase) with microsyringe and inject in the film, with sealing the membrane closure mouth of pipe.Extraction equipment is put into oscillator, and the speed oscillation with 600r/min under the room temperature extracts 90min.The extraction back is drawn and is all received phase, is transferred in the sample introduction bottle, treats that HPLC surveys.Take by weighing 1.01g sodium heptanesulfonate and 1.05g citric acid, be dissolved in water and be settled to 500mL, make ion-pairing agent.
The HPLC model is Tianjin, island LC-20A, and detecting device is diode array detector SPD-M20A.Chromatographic column is DiamonsilTM (two generations of a diamond) C18 chromatographic column, column length 250mm, internal diameter 4.6mm, particle diameter 5 μ m.The moving phase condition be acetonitrile/ion-pairing agent (10/90, V/V), flow velocity 1.2mL/min, 30 ℃ of column temperatures detect wavelength 240nm.With its response peak area and retention time mapping on chromatogram.
Testing result is as shown in table 4:
Blank and the mark-on measuring result of table 4 milk powder matrix
Annotate: RSD is the relative standard deviation of peak area.
By the result as can be known, under this chromatographic condition, the noiseless thing of milk powder matrix influences the qualitative and quantitative of melamine, and average recovery of standard addition is 75.6%, and relative standard deviation is 12.1%.Illustrate that this method can be used for the mensuration of melamine in the milk powder.
Detect the liquid milk sample:
Preparation 1mg/mL melamine standard solution.Accurately measure 8mL liquid milk sample, the mark-on group adds melamine standard solution 16 μ L, 4 μ L, 0.8 μ L (i.e. interpolation level is respectively 2 μ g/mL, 0.5 μ g/mL, 0.1 μ g/mL), three samples of every group of parallel processing respectively.Extracting tube was immersed in the n-octyl alcohol ultrasonic 10 seconds, takes out the back and sops up unnecessary n-octyl alcohol with lens wiping paper.Extracting tube is inserted in the sample bottle, draw 20 μ L distilled water (reception phase) with microsyringe and inject in the film, with sealing the membrane closure mouth of pipe.Extraction equipment is put into oscillator, and the speed oscillation with 600r/min under the room temperature extracts 90min.The extraction back is drawn and is all received phase, is transferred in the sample introduction bottle, treats that HPLC surveys.Take by weighing 1.01g sodium heptanesulfonate and 1.05g citric acid, be dissolved in water and be settled to 500mL, make ion-pairing agent.
The liquid condition of extraction gained as shown in Figure 7 from emulsion.
The HPLC model is Tianjin, island LC-20A, and detecting device is diode array detector SPD-M20A.Chromatographic column is DiamonsilTM (two generations of a diamond) C18 chromatographic column, column length 250mm, internal diameter 4.6mm, particle diameter 5 μ m.The moving phase condition be acetonitrile/ion-pairing agent (10/90, V/V), flow velocity 1.2mL/min, 30 ℃ of column temperatures detect wavelength 240nm.With its response peak area and retention time mapping on chromatogram.
Experimental result is as shown in table 5:
The horizontal measuring result of the different interpolations of table 5
Annotate: RSD is the relative standard deviation of peak area.
As shown in Figure 7, extraction obtains the sample solution of clear from the emulsion of muddiness, can be applied directly to HPLC and detect, and need not further 0.45 μ m filter membrane and purifies.
Result by table 5 shows: in adding concentration 0.1 μ g/mL～2 μ g/mL concentration ranges, the method recovery is between 72.10%～100.5%, and relative standard deviation is less than 5%.Illustrate that this method recovery is high and stable, repeatability is strong, the precision height.
This method is under 0.1 μ g/mL (being 0.1ppm) interpolation level, and the object melamine still has higher response.In the existing national standard method (GB/T22388-2008), high performance liquid chromatography quantitatively be limited to 2mg/kg, i.e. 2ppm.Illustrate that this method carries out sample pre-treatments by liquid-phase microextraction method, the effect that can reach the enrichment object and remove chaff interference is compared with general high-efficiency liquid chromatography method for detecting, can reach lower detectability.It is highly sensitive to illustrate that this method has, the advantage that detectability is low.
The quantitative limit that liquid milk detects is measured:
Preparation 0.1mg/mL melamine standard solution.Accurately measure 8mL liquid milk sample, the mark-on group adds melamine standard solution 4 μ L (i.e. interpolation level is respectively 0.05 μ g/mL), and blank is organized not mark-on, three samples of every group of parallel processing.Extracting tube was immersed in the n-octyl alcohol ultrasonic 10 seconds, takes out the back and sops up unnecessary n-octyl alcohol with lens wiping paper.Extracting tube is inserted in the sample bottle, draw 20 μ L distilled water (reception phase) with microsyringe and inject in the film, with sealing the membrane closure mouth of pipe.Extraction equipment is put into oscillator, and the speed oscillation with 600r/min under the room temperature extracts 90min.The extraction back is drawn and is all received phase, is transferred in the sample introduction bottle, treats that HPLC surveys.Take by weighing 1.01g sodium heptanesulfonate and 1.05g citric acid, be dissolved in water and be settled to 500mL, make ion-pairing agent.
The HPLC model is Tianjin, island LC-20A, and detecting device is diode array detector SPD-M20A.Chromatographic column is DiamonsilTM (two generations of a diamond) C18 chromatographic column, column length 250mm, internal diameter 4.6mm, particle diameter 5 μ m.The moving phase condition be acetonitrile/ion-pairing agent (10/90, V/V), flow velocity 1.2mL/min, 30 ℃ of column temperatures detect wavelength 240nm.With its response peak area and retention time mapping on chromatogram.
Experimental result is as shown in table 6:
Table 6 quantitative limit measuring result
Annotate: RSD is the relative standard deviation of peak area.
The result shows, this method quantitatively be limited to 0.05 μ g/mL.
Claims (10)
1. a method that detects melamine in raw milk and the dairy products comprises the detection of sample pre-treatments and high performance liquid chromatography, it is characterized in that: described sample pre-treatments adopts hollow-fibre membrane three-phase liquid-phase micro-extraction to carry out.
2. the method for melamine in detection raw milk as claimed in claim 1 and the dairy products is characterized in that described hollow-fibre membrane three-phase liquid-phase micro-extraction is to place testing sample solution to extract at the tunica fibrosa coating that will contain organic phase and water; Described organic phase is adsorbed in the hole wall of tunica fibrosa, and described water is coated on the inside of tunica fibrosa.
3. the method for melamine in detection raw milk as claimed in claim 2 and the dairy products is characterized in that described organic phase is n-octyl alcohol or toluene; Described tunica fibrosa is Kynoar tunica fibrosa, polyethylene fibre film or polypropylene screen tunica fibrosa.
4. the method for melamine in detection raw milk as claimed in claim 2 and the dairy products is characterized in that described tunica fibrosa coating is the tubule shape, its wall thickness 100～300 μ m, internal diameter 1.2～10mm, aperture 0.1～0.3 μ m.
5. the method for melamine in detection raw milk as claimed in claim 2 and the dairy products is characterized in that the described extraction time is 30～120min.
6. the method for melamine in detection raw milk as claimed in claim 2 and the dairy products is characterized in that described extraction process is to carry out under the rotating speed of 500～1200rpm.
7. the method for melamine in detection raw milk as claimed in claim 1 or 2 and the dairy products is characterized in that described raw milk and dairy products are that liquid milk, milk powder, cheese, cream or chocolate-like contain dairy products.
8. the method for melamine in detection raw milk as claimed in claim 7 and the dairy products is characterized in that described milk powder becomes testing sample by dissolving; Cheese, cream or chocolate-like contain dairy products becomes testing sample through dissolving again by after pulverizing.
9. one kind is used for the sample pre-treatments device of method according to claim 1, comprise the bottle with cover that testing sample solution is housed, it is characterized in that the lid below is hung with the tunica fibrosa coating that contains organic phase and water, organic phase wherein is adsorbed in the hole wall of tunica fibrosa; Described coating at least a portion is soaked in the sample solution.
10. the preparation method of a sample pre-treatments device as claimed in claim 9 is characterized in that may further comprise the steps:
(1) will clutch sealing by the end that hollow-fibre membrane constitutes, the other end links to each other with suction nozzle, becomes extracting tube, and the hollow-fibre membrane end is the extracting tube bottom, and plastics suction nozzle end is the extracting tube top;
(2) extracting tube is immersed in the organic phase solution, take out ultrasonic back;
(3) top of the extracting tube of step (2) gained is fixed in the bottle cap of sample bottle, when covering bottle cap, at least a portion of tunica fibrosa will be soaked in the sample solution in the sample bottle; And the interface of tunica fibrosa and suction nozzle is positioned on the sample solution.
(4) inject water from the extracting tube top toward extracting tube, it is full of or the part be full of the hollow fiber membrane portions, seal the extracting tube top again.
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