CN108445118B - Method for rapidly extracting and detecting sucralose in food - Google Patents
Method for rapidly extracting and detecting sucralose in food Download PDFInfo
- Publication number
- CN108445118B CN108445118B CN201810228640.6A CN201810228640A CN108445118B CN 108445118 B CN108445118 B CN 108445118B CN 201810228640 A CN201810228640 A CN 201810228640A CN 108445118 B CN108445118 B CN 108445118B
- Authority
- CN
- China
- Prior art keywords
- sample
- sucralose
- acetonitrile
- solution
- extracted
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a method for quickly extracting and detecting sucralose in food, which comprises the following steps: (1) preparing a solution to be detected; (2) preparing a standard working solution and a standard working curve; (3) and (2) taking acetonitrile and water as mobile phases, separating the solution to be detected prepared in the step (1) and the standard working solution prepared in the step (3) through a C18 chromatographic column, eluting, detecting eluent by adopting a high performance liquid chromatograph equipped with an evaporation light detector, qualitatively judging sucralose according to the retention time of the sample and the standard substance, and drawing an index correction curve on the peak area for quantification. The method adopts specific extraction solvent and auxiliary extraction solvent during pretreatment, can effectively extract the sucralose in the food into acetonitrile, has obvious effect of removing impurities such as pigment, protein and the like in the sample, and does not interfere with the qualitative and quantitative determination of the sucralose.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a rapid extraction method and a content detection method of sucralose in food.
Background
Sucralose is a non-nutritional strong sweetener prepared by chlorination of sucrose as a raw material, and has the Chinese alias: sucralose; sucrose crystals; trichlorogalactose. The chemical name is as follows: 4,1',6' -trichloro-4, 1',6' -trideoxygalactosucrose CAS No.56038-13-2, molecular formula C12H19Cl3O8Molecular weight 397.064, is a white powder product. The sweetness of the sucralose is 600 times that of the sucrose, is very easy to dissolve in water, and has the advantages of pure sweetness, good chemical stability, unchanged sweetness at high temperature, more stable sweetness, no toxic or side effect, almost no absorption in a human body, zero caloric value, being a sweet substitute for a diabetic patient and the like. The sucralose is widely applied to processed foods such as soy sauce, beverages, dairy products, alcoholic beverages, jam and the like, and is one of the most characteristic advantages among various sweeteners allowed to be used at home and abroad.
The detection standard of sucralose in food at present comprises national mandatory standard GB 22255-. GB 22255-. The DBS52/007-2014 standard specifies a liquid chromatography-mass spectrometry detection method for sodium cyclamate, saccharin sodium, acesulfame potassium and sucralose in white spirit, and the pretreatment method adopts direct filtration and then detection on a machine. In addition, the extraction method of sucralose in foods reported in domestic literature is mainly a solid phase extraction method, a direct dilution method or an extraction method directly adopting GB22255-2014, wherein the solid phase extraction method is mainly used, and the used solid phase extraction column comprises an HLB column, a neutral alumina column and the like. GB22255-2014, DBS52/007-2014 and the pretreatment methods reported in the literature have no obvious pigment removal effect on samples containing a large amount of melanin such as soy sauce, sauce products and the like, and when the solid-phase extraction pretreatment method is adopted, the sucralose and most of the pigments are retained on the solid-phase extraction column and eluted along with the eluent, so that the purposes of removing the pigments and purifying the samples cannot be achieved. The direct dilution method is only applicable to samples with low or no pigment content. The presence of large amounts of pigments can easily damage the chromatographic column during the detection process, contaminate the detector, and may even affect the qualitative and quantitative properties.
Patent 03805527.9 "extraction method for purifying sucralose" is to separate high-purity sucralose from industrially synthesized sucralose from a complex mixture, and the method is to extract and crystallize sucralose in the complex mixture for multiple times by using ethyl acetate as an organic extraction solvent, so as to remove impurities in a sucralose product to the maximum extent and obtain high-purity sucralose.
The literature reports that sucralose detection methods include high performance liquid chromatography, ion chromatography, liquid-mass spectrometry, capillary electrophoresis, chemical titration, and the like. GB22255-2014 adopts high performance liquid phase-evaporative light scattering or differential refractive detection, and DBS52/007-2014 adopts liquid chromatography-tandem mass spectrometry. The high performance liquid chromatography evaporative light scattering or differential refraction detection method has strong universality and low cost, but the detection method of sucralose to be adopted subsequently is different due to different pretreatment technologies.
The prior sucralose detection pretreatment technology comprises a solid phase extraction method and a direct dilution method, and the methods have unobvious pigment removal effect on samples containing pigments, especially samples containing a large amount of pigments such as soy sauce, sauce products and the like. According to the solid-phase extraction methods such as an HLB (hydrophile-lipophile balance) column and a neutral alumina column, the sucralose and most of the pigment are retained on the solid-phase extraction column and eluted along with the eluent, so that the pigment and the sucralose cannot be separated, the pigment cannot be effectively removed, and the sample is purified; the direct dilution method is only applicable to samples with low or no pigment content. The presence of large amounts of pigments can easily damage the chromatographic column during the detection process, contaminate the detector, and may even affect the qualitative and quantitative properties. Therefore, there is a need to explore new pretreatment methods and the parameters of sucralose testing methods and instruments used in conjunction therewith.
Disclosure of Invention
The invention aims to provide a method for quickly extracting sucralose from food and a detection method, wherein a specific extraction solvent and an auxiliary extraction solvent are adopted during pretreatment, the sucralose in the food can be effectively extracted into acetonitrile, the removal effect of impurities such as pigments, proteins and the like in a sample is very obvious, and the coexisting impurities do not interfere the qualitative and quantitative determination of the sucralose through the separation of a chromatographic column.
The above object of the present invention is achieved by the following technical solutions: a method for rapidly extracting and detecting sucralose in food comprises the following steps:
(1) preparing a solution to be detected: selecting a sample containing protein and/or fat, adding ultrapure water, shaking up to obtain a sample to be extracted, adding acetonitrile into the sample to be extracted, carrying out vortex, respectively adding zinc acetate and potassium ferrocyanide solution, then sequentially adding sodium chloride and anhydrous magnesium sulfate, carrying out centrifugation after vortex, taking an upper acetonitrile layer, repeatedly extracting a lower sample to be extracted with acetonitrile, combining the acetonitrile layers, carrying out rotary evaporation to dryness, dissolving with ultrapure water, adding n-hexane, carrying out vortex, standing, taking a lower aqueous phase out of a water system membrane, and obtaining a solution to be detected;
or selecting sauce, sauce products, vinegar, soy sauce and beverage samples, adding ultrapure water, shaking up to obtain a sample to be extracted, adding acetonitrile into the sample to be extracted, carrying out vortex, adding sodium chloride and anhydrous magnesium sulfate, carrying out centrifugation after vortex, taking an upper acetonitrile layer, repeatedly extracting a lower sample to be extracted with acetonitrile, combining the acetonitrile layers, carrying out rotary evaporation to dryness, dissolving with ultrapure water, adding n-hexane, carrying out vortex, standing, taking a lower aqueous phase and passing through a water system membrane to obtain a solution to be detected;
(2) adding ultrapure water into a sucralose standard substance for dissolving, uniformly mixing, and preparing a standard working solution and a standard working curve by using the ultrapure water;
(3) and (2) taking acetonitrile and water as mobile phases, separating the solution to be detected prepared in the step (1) and the standard working solution prepared in the step (3) through a C18 chromatographic column, eluting, detecting eluent by adopting a high performance liquid chromatograph equipped with an evaporation light detector, qualitatively judging sucralose according to retention time of a sample and a standard substance, and quantitatively determining the sucralose according to an index correction curve drawn according to a peak area.
In the method for rapidly extracting and detecting the sucralose in the food, the steps of:
the protein and/or fat-containing sample, the sauce and sauce product, the vinegar, the soy sauce and the beverage sample in the step (1) are solid samples, semi-solid samples or liquid samples; the mass-volume ratio of the solid sample to the semi-solid sample to the ultrapure water is 1-2 g: 10-20 mL, wherein the mass-to-volume ratio of the liquid sample to the ultrapure water is 2-5 g: 8-5 mL.
The protein and/or fat sample can be a solid sample, a semi-solid sample or a liquid sample, wherein the solid sample is crushed, the semi-solid sample is crushed or stirred, and the liquid sample is shaken up and then diluted by water to obtain a uniform sample to be detected for use, so that the sucralose in the sample is released into the water.
The sauce and sauce products, vinegar, soy sauce and beverage samples are liquid samples.
When the sample is protein and/or fat, acetonitrile is used as an extraction reagent, and potassium ferrocyanide, zinc acetate, sodium chloride and anhydrous magnesium sulfate are used as auxiliary extraction reagents, so that the extraction efficiency and the extraction recovery rate of the sucralose can be improved.
When the samples are sauce, sauce products, vinegar, soy sauce and beverage samples, acetonitrile is used as an extraction reagent, and sodium chloride and anhydrous magnesium sulfate are used as auxiliary extraction reagents, so that the extraction efficiency and the extraction recovery rate of the sucralose can be improved.
When the sample is sauce, sauce products, vinegar, soy sauce and beverage, the extraction, purification and detection methods are consistent with those of protein and/or fat samples except that the zinc acetate solution and the potassium ferrocyanide solution are not added.
In the step (1), the volume ratio of the sample to be extracted to the acetonitrile is 1: 1 to 2.5.
In the invention, the sucralose has stronger polarity and is easily dissolved in organic reagents such as methanol, acetonitrile, ethyl acetate and the like. Acetonitrile and ethyl acetate are mixed and dissolved with water, and then are obviously layered under the salting-out action, thereby being beneficial to purifying and concentrating samples. The methanol and the water can not be separated under the salting-out action after being mixed and dissolved, and the purposes of purification and concentration can not be achieved. By refining and comparing the extraction effects of acetonitrile and ethyl acetate, the acetonitrile has a better effect of dissolving sucralose due to strong polarity, has a better extraction effect on sucralose than that of ethyl acetate, has a higher recovery rate, but has more extracted impurities than that of ethyl acetate, and does not interfere with the qualitative and quantitative analysis of sucralose after the impurities are separated by a chromatographic column, so that the acetonitrile is used as an organic extraction reagent.
The volume ratio of the zinc acetate solution to the potassium ferrocyanide solution to the sample to be extracted in the step (1) is 1-2: 10-20, wherein the volume ratio of the zinc acetate solution to the potassium ferrocyanide solution is 1: 1, wherein the mass concentration of the zinc acetate solution is 200g/L, and the mass concentration of the potassium ferrocyanide solution is 100 g/L.
The volume of the zinc acetate solution and the potassium ferrocyanide solution added in the step (1) is determined according to the protein content of the sample, and generally the ratio of the sum of the added volumes of the zinc acetate solution and the potassium ferrocyanide solution to the volume of the sample to be extracted is 1-2: 10-20, wherein the volume ratio of the zinc acetate solution to the potassium ferrocyanide solution is 1: 1.
the zinc acetate and potassium ferrocyanide solution added in the step has the functions of precipitating protein and purifying the sample aiming at the sample containing higher protein, and the addition amount of the zinc acetate and the potassium ferrocyanide solution is based on the effective precipitation of the protein.
The method has the advantages that the sucralose has strong polarity and is very soluble in water, the sodium chloride can play a salting-out role and acts together with the anhydrous magnesium sulfate to remove water in an organic phase, the solubility of the sucralose in water is reduced, the solubility of the sucralose in a polar solvent acetonitrile is increased by utilizing the principle of similarity and intermiscibility, and thus the extraction recovery rate is improved.
Therefore, preferably, the mass-to-volume ratio of the sodium chloride to the sample to be extracted in step (1) of the present invention is 3 to 5 g: 10-20 mL, wherein the mass-volume ratio of the anhydrous magnesium sulfate to the sample to be extracted is 1-2 g: 10-20 mL, wherein the mass ratio of the sodium chloride to the anhydrous magnesium sulfate is 1-3: 2 to 5.
When the sample to be extracted in the step (1) is repeatedly extracted by acetonitrile, the distribution ratio difference exists in liquid-liquid extraction, and the single extraction recovery rate cannot meet the detection requirement, so that the pretreatment method can ensure that the extraction recovery rate reaches over 80 percent after 2 times of extraction.
Therefore, preferably, the repeated extraction of the lower layer sample to be extracted with acetonitrile in step (1) of the present invention means that the repeated extraction of the lower layer sample to be extracted with acetonitrile is performed twice or more.
Combining acetonitrile layers in the step (1), carrying out rotary evaporation to dryness under the conditions of water bath at 46-50 ℃ and negative pressure, dissolving with ultrapure water to obtain 1-4 mL of a dissolved sample, and adding n-hexane, wherein the volume ratio of the n-hexane to the dissolved sample is 1-5: 1.
the acetonitrile has high rotary evaporation speed at 46-50 ℃ and can avoid liquid bumping, and the n-hexane is added in the invention to remove a small amount of residual grease and fat-soluble pigment, thereby further removing impurities and purifying.
The mobile phase adopted by the high performance liquid chromatograph in the step (3) is acetonitrile and water, and the volume ratio of the acetonitrile to the water is 14: 86-20: 80, the chromatographic column is a C18 chromatographic column, the specification is 250mm multiplied by 4.6mm, the particle size is 5 mu m, the temperature of the chromatographic column is room temperature, the sample injection amount is 10-50 mu L, the flow rate of a mobile phase is 1.0mL/min, the ELSD evaporation temperature is 40-60 ℃, the atomizing gas is nitrogen, and the gas pressure is 350-420 KPa.
The method adopts an isocratic elution mode, the ratio of acetonitrile and water of a fixed mobile phase does not change along with time, the flow rate of the mobile phase is 1.0mL/min, the temperature of a chromatographic column is room temperature, the chromatographic column is matched with 250mm multiplied by 4.6mm, the particle size is 5 mu m, and the chromatographic column realizes the effective separation of sucralose and impurities. The ELSD evaporation temperature is 40-60 ℃, the gas pressure is 350-420 KPa, the atomization of the mobile phase loaded with the sucralose is ensured, the interference is reduced, and the detection sensitivity is improved.
Compared with the prior art, the invention has the following advantages:
(1) the method of the invention is applicable to a wide range of foods, including but not limited to protein and/or fat samples, foods such as sauce and soy products, vinegar, soy sauce, beverage samples and the like;
(2) the method adopts acetonitrile as an extraction solvent and potassium ferrocyanide, zinc acetate, sodium chloride and anhydrous magnesium sulfate as auxiliary extraction reagents, and has no relevant literature report or relevant detection standard at home and abroad at present, so that the method has obvious innovation;
(3) the method adopts zinc acetate and potassium ferrocyanide solution as precipitating agents for a sample with high protein content, and combines salting out purification and acetonitrile extraction by sodium chloride and anhydrous magnesium sulfate to remove protein and improve the extraction recovery rate;
(4) the method adopts sodium chloride to ensure that the water phase in a sample to be extracted is in a saturated state, reduces the solubility of an organic phase and sucralose in water, plays a role of salting out, and anhydrous magnesium sulfate absorbs water but does not aim at completely absorbing the water in the sample, so that the aim of locking the water in the organic phase in the water phase is fulfilled to remove the water in the organic phase, and the sodium chloride salting out effect is cooperated to improve the extraction effect of acetonitrile on the sucralose, so that the recovery rate reaches more than 80%;
(5) the method takes acetonitrile with strong polarity as an extraction solvent, can effectively extract sucralose in food into the acetonitrile, adopts zinc acetate and potassium ferrocyanide solution to assist in precipitating protein, adopts salting out and purification of sodium chloride and anhydrous magnesium sulfate to effectively extract sucralose easily soluble in water into the acetonitrile, has obvious pigment removal effect, and achieves a sucralose recovery rate of more than 80 percent, and the established pretreatment method has no literature or related national standard reports, solves the problems of low sucralose extraction recovery rate and poor pigment removal effect in various foods, and has purification effects incomparable with solid-phase extraction and direct dilution methods;
(6) the method adopts acetonitrile as an extraction solvent, and potassium ferrocyanide, zinc acetate, sodium chloride and anhydrous magnesium sulfate as auxiliary extraction reagents, and aims to extract most of sucralose in food into the acetonitrile through the comprehensive action of the extraction solvent and the auxiliary extraction reagents.
Drawings
FIG. 1 is a chromatogram of a sucralose standard concentration of 1000. mu.g/mL in example 1;
FIG. 2 is a chromatogram of a sample of white soy sauce in example 1;
FIG. 3 is a chromatogram obtained by extracting 2 times with acetonitrile a sample of white soy sauce obtained in example 1 or 3, to which a standard substance (100mg/kg) was added;
FIG. 4 is a chromatogram obtained by extracting 2 times with ethyl acetate a sample of hollow white soy sauce obtained in example 3 with a standard (100 mg/kg);
FIG. 5 is a chromatogram of the sucralose-free milk powder of example 2;
FIG. 6 is a chromatogram of the addition of a standard (40mg/kg) to a sample of the hollow white milk powder of example 2;
FIG. 7 is an exponential fit standard curve for sucralose in examples 1-2.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the scope of the invention as claimed is not limited to the following embodiments.
The apparatus and equipment used in the invention:
(1) shimadzu high performance liquid chromatograph (LC-20A) equipped with evaporative light scattering detector (ELSD-LT II);
(2) SIGMA high-speed freezing centrifuge 3-18K;
(3) an IKA vortex mixer;
(4) EYELA rotary evaporator;
(5) an electronic balance: the dosages are 0.01g and 0.0001g respectively;
the reagents used in the present invention:
(1) acetonitrile and ethyl acetate used in the sample extraction process are analytically pure;
(2) acetonitrile used in a mobile phase is chromatographically pure;
(3) the water used in the detection process is ultrapure water.
Example 1
In this example, soy sauce was used as a sample for detection.
First, pretreatment step
1.1 extraction and purification
Weighing 5g (accurate to 0.01g) of uniform soy sauce sample, placing the uniform soy sauce sample into a 50mL plastic centrifuge tube, adding 5mL of ultrapure water, shaking up to obtain a sample to be extracted, adding 20mL of acetonitrile into the sample to be extracted, carrying out vortex for 1min, sequentially adding 5g of sodium chloride and 2g of anhydrous magnesium sulfate, immediately and violently carrying out vortex for 1min to thicken the sample, centrifuging for 2min at 9000r/min, and transferring the supernatant (acetonitrile layer) into a 125mL heart-shaped bottle. Repeatedly extracting a sample to be extracted in a centrifuge tube for 1 time by using 20mL of acetonitrile, vortexing for 1min, centrifuging for 2min at 9000r/min, transferring the acetonitrile into a heart-shaped bottle, rotatably evaporating to dryness in a water bath at 46-50 ℃, dissolving with 1.0mL of ultrapure water, vortexing for 30s, adding 1.0mL of n-hexane, vortexing, standing for layering, taking a lower-layer water phase, passing through a 0.45-micron water system membrane, and placing in a sample inlet bottle to obtain a solution to be detected.
1.2 preparation of Standard stock solutions
Precisely weighing about 15mg (accurate to 0.1mg) of sucralose standard, placing the sucralose standard in a 10mL volumetric flask, adding ultrapure water, shaking up gently, then using the ultrapure water to fix the volume to 10mL, and shaking up to obtain a standard substance stock solution of 1500 mu g/mL.
1.3 preparation of Standard working Curve
Taking a proper volume of sucralose standard stock solution, and diluting the sucralose standard stock solution with ultrapure water step by step to prepare a standard working curve, wherein the preparation concentration range is 20-1500 mu g/mL.
Second, liquid phase chromatographic conditions
Volume ratio of mobile phase: acetonitrile: 14 parts of water: 86, isocratic elution mode; a chromatographic column: agilent ZORBAX SB-C18(250 mm. times.4.6 mm, 5 μm); flow rate: 1.0 mL/min; column temperature: room temperature; sample introduction amount: 20 mu L of the solution; the evaporation temperature of the solvent of the evaporation light detector is 40-60 ℃, the atomizing gas is nitrogen, and the pressure of the nitrogen is 350-420 KPa.
Under the chromatographic conditions of the example, the sucralose has better separation degree and sensitivity from impurities, the peak shape is better, the retention time of the sucralose is 12.1min, and the chromatogram of the standard (1000. mu.g/mL) is shown in FIG. 1.
Third, sample determination
And (3) sucking 20 mu L of sample solution by an automatic sample injector of the device under the set liquid phase condition, detecting by high performance liquid chromatography-evaporative light scattering, and indicating that the sample contains the sucralose when a chromatographic peak consistent with the retention time of the sucralose standard substance exists in the sample. If the mass concentration of the target in the solution to be measured exceeds the upper limit of the linear range of the standard curve, the sample to be measured needs to be diluted properly so as to be measured in the linear range of the standard curve.
Four, linear equation, method detection limit
The detection limit of the method is inspected by adding a standard substance into a soy sauce sample without sucralose. The linear range of exponential fitting of the sucralose is 20-1500 mug/mL, and the linear equation of exponential fitting is that Y is 0.745X EXP (-3.826), R2The detection limit of the method is 1mg/kg, the quantification limit is 3mg/kg, and the detection limit (2.5mg/kg) and the quantification limit (7.5mg/kg) are lower than those of GB 22255-. Index fitting standardThe quasi-curve is shown in FIG. 7.
Fifthly, the recovery rate and precision of the added standard
And (3) adopting a mode of adding a standard substance into a soy sauce sample without sucralose to examine the recovery rate and precision of the method. Three levels of standard substances are respectively added, the addition levels are 4, 40 and 100mg/kg, and the recovery rate is 83.2-95.4%. The specific recovery rate and precision are shown in Table 1.
The chromatogram of the blank soy sauce sample (without sucralose) is shown in FIG. 2, and the chromatogram of the blank soy sauce sample with standard (100mg/kg) is shown in FIG. 3.
TABLE 1 recovery and precision
Sixthly, quantitative calculation
And (4) carrying out exponential fitting on a standard curve, and quantifying by an external standard method.
The calculation formula is as follows:
X＝C×V×f/m
wherein:
x: content of sucralose in sample (mg/kg)
C: the concentration of the component to be measured (. mu.g/mL) was obtained from the standard working curve
m: sampling quality (g)
V: constant volume (mL)
f: dilution factor
The result is to retain three significant digits.
Seventhly, detecting actual samples
The soy sauce samples marked with sucralose as detection objects are purchased in the market for 20 batches, and the content of the sucralose detected by the method in the embodiment is 0.0742-0.201 g/kg.
Example 2
In this example, goat milk powder containing protein and fat was used as the test sample.
First, pretreatment step
1.1 extraction and purification
Weighing 1g (accurate to 0.01g) of uniform milk powder sample, placing the uniform milk powder sample into a 50mL plastic centrifuge tube, adding 10mL of ultrapure water, shaking up to obtain a sample to be extracted, adding 20mL of acetonitrile into the sample to be extracted, carrying out vortex for 1min, adding 1mL of each of zinc acetate and potassium ferrocyanide solution, wherein the concentration of the zinc acetate solution is 200g/L, the concentration of the potassium ferrocyanide solution is 100g/L, sequentially adding 5g of sodium chloride and 2g of anhydrous magnesium sulfate, carrying out vortex for 1min immediately and violently, thickening the sample, carrying out centrifugation for 2min at 9000r/min, and transferring the supernatant (acetonitrile layer) into a 125mL heart-shaped bottle. Repeatedly extracting a sample to be extracted in a centrifuge tube for 1 time by using 20mL of acetonitrile, vortexing for 1min, centrifuging for 2min at 9000r/min, transferring the acetonitrile into a heart-shaped bottle, rotatably evaporating to dryness in a water bath at 46-50 ℃, dissolving with 1.0mL of ultrapure water, vortexing for 30s, adding 1.0mL of n-hexane, vortexing, standing for layering, taking a lower-layer water phase, passing through a 0.45-micron water system membrane, and placing in a sample inlet bottle to obtain a solution to be detected.
1.2 preparation of Standard stock solutions
Precisely weighing about 15mg (accurate to 0.1mg) of sucralose standard, placing the sucralose standard in a 10mL volumetric flask, adding ultrapure water, shaking up gently, then using the ultrapure water to fix the volume to 10mL, and shaking up to obtain a standard substance stock solution of 1500 mu g/mL.
1.3 preparation of Standard working Curve
Taking a proper volume of sucralose standard stock solution, and diluting the sucralose standard stock solution with ultrapure water step by step to prepare a standard working curve, wherein the preparation concentration range is 20-1500 mu g/mL.
Second, liquid phase chromatographic conditions
Volume ratio of mobile phase: acetonitrile and water in 14:86, and an isocratic elution mode; a chromatographic column: agilent ZORBAX SB-C18(250 mm. times.4.6 mm, 5 μm); flow rate: 1.0 mL/min; column temperature: room temperature; sample introduction amount: 20 mu L of the solution; the evaporation temperature of the solvent of the evaporation light detector is 40-60 ℃, the atomizing gas is nitrogen, and the pressure of the nitrogen is 350-420 KPa.
The milk powder sample contains a large amount of impurities such as protein, saccharides and the like, under the pretreatment method and the chromatographic separation condition of the embodiment, the sucralose and the impurities have better separation degree and sensitivity, the peak shape is better, the blank sample chromatogram shows that no interference of other impurity peaks exists at the position of the sucralose chromatogram peak, the blank sample chromatogram is shown in figure 5, and the labeled sample chromatogram is shown in figure 6.
Third, sample determination
And (3) sucking 20 mu L of sample solution by an automatic sample injector of the device under the set liquid phase condition, detecting by high performance liquid chromatography-evaporative light scattering, and indicating that the sample contains the sucralose when a chromatographic peak consistent with the retention time of the sucralose standard substance exists in the sample. If the mass concentration of the target in the solution to be measured exceeds the upper limit of the linear range of the standard curve, the sample to be measured needs to be diluted properly so as to be measured in the linear range of the standard curve.
Four, linear equation, method detection limit
The detection limit of the method is inspected by adding a standard substance into a milk powder sample without sucralose. The linear range of exponential fitting of the sucralose is 20-1500 mug/mL, and the linear equation of exponential fitting is that Y is 0.745X EXP (-3.826), R2The detection limit of the method is 2mg/kg, the quantification limit is 6mg/kg, and the detection limit (2.5mg/kg) and the quantification limit (7.5mg/kg) are lower than those of GB 22255-. The standard curve of the exponential fit is shown in FIG. 7.
Fifthly, the recovery rate and precision of the added standard
And (3) observing the recovery rate and precision of the method by adding a standard substance into a milk powder sample without sucralose. Three levels of standard substances are respectively added, the addition levels are 4, 40 and 100mg/kg, and the recovery rate is 81.2-92.3%. The specific recovery rate and precision are shown in Table 2.
The chromatogram of the blank milk powder sample (without sucralose) is shown in FIG. 5, and the chromatogram of the blank sample with the standard (40mg/kg) is shown in FIG. 6.
TABLE 2 recovery and precision
Sixthly, quantitative calculation
The quantitative calculation was in agreement with that of example 1.
Seventhly, detecting actual samples
The milk powder samples purchased in the market were used as the test objects in 10 batches, and no sucralose was detected by the test method in this example.
Example 3
In this example, soy sauce was used as a test sample, and the effect of acetonitrile and ethyl acetate on the sucralose extraction recovery rate was compared.
The blank soy sauce samples were supplemented with three levels of 4, 40, 100mg/kg standards and measured 6 times per level (n-6). Acetonitrile is taken as an organic extraction solvent, and the operation is carried out according to the implementation steps described in the embodiment 1; the acetonitrile of example 1 was then replaced with ethyl acetate as the extraction solvent and the remaining steps were carried out as described in example 1. The recovery and precision data obtained are shown in table 3. FIG. 3 is a chromatogram of a blank soy sauce sample added with a standard (100mg/kg) and extracted with acetonitrile for 2 times; FIG. 4 is a chromatogram of a blank soy sauce sample after adding a standard (100mg/kg) and extracting with ethyl acetate for 2 times. As can be seen from the data in Table 3, the recovery of acetonitrile extraction is significantly higher than that of ethyl acetate. Comparing fig. 3 and fig. 4, it can be seen that the impurities extracted by acetonitrile are more and the impurities extracted by ethyl acetate are less, but the impurity peaks extracted by both do not interfere with the sucralose chromatographic peak, the comprehensive recovery rate is high, and acetonitrile is selected as the organic extraction solvent of the present invention.
TABLE 3 recovery and precision
The present invention has been described above by referring to a part of specific embodiments, and it should be noted that the above-mentioned specific embodiments are only used for further description of the present invention and do not represent a limitation to the scope of the present invention. Other insubstantial modifications and adaptations of the present invention can be made without departing from the scope of the present invention.
Claims (7)
1. A method for rapidly extracting and detecting sucralose in food is characterized by comprising the following steps:
(1) preparing a solution to be detected: selecting a sample containing protein and/or fat, adding ultrapure water, shaking up to obtain a sample to be extracted, adding acetonitrile into the sample to be extracted, carrying out vortex, respectively adding zinc acetate and potassium ferrocyanide solution, then sequentially adding sodium chloride and anhydrous magnesium sulfate, carrying out centrifugation after vortex, taking an upper acetonitrile layer, repeatedly extracting a lower sample to be extracted with acetonitrile, combining the acetonitrile layers, carrying out rotary evaporation to dryness, dissolving with ultrapure water, adding n-hexane, carrying out vortex, standing, taking a lower aqueous phase out of a water system membrane, and obtaining a solution to be detected;
or selecting sauce, sauce products, vinegar, soy sauce and beverage samples, adding ultrapure water, shaking up to obtain a sample to be extracted, adding acetonitrile into the sample to be extracted, carrying out vortex, adding sodium chloride and anhydrous magnesium sulfate, carrying out centrifugation after vortex, taking an upper acetonitrile layer, repeatedly extracting a lower sample to be extracted with acetonitrile, combining the acetonitrile layers, carrying out rotary evaporation to dryness, dissolving with ultrapure water, adding n-hexane, carrying out vortex, standing, taking a lower aqueous phase and passing through a water system membrane to obtain a solution to be detected;
(2) adding ultrapure water into a sucralose standard substance for dissolving, uniformly mixing, and preparing a standard working solution and a standard working curve by using the ultrapure water;
(3) separating the solution to be detected prepared in the step (1) and the standard working solution prepared in the step (2) by using acetonitrile and water as mobile phases through a C18 chromatographic column, eluting, detecting eluent by using a high performance liquid chromatograph equipped with an evaporation light detector, qualitatively judging sucralose according to retention time of a sample and a standard substance, and drawing an index correction curve according to a peak area to quantify the sucralose;
the mobile phase adopted by the high performance liquid chromatograph in the step (3) is acetonitrile and water, and the volume ratio of the acetonitrile to the water is 14: 86-20: 80, the chromatographic column is an Agilent ZORBAX SB-C18 chromatographic column, the specification is 250mm multiplied by 4.6mm, the particle size is 5 mu m, the temperature of the chromatographic column is room temperature, the sample injection amount is 10-50 mu L, the flow rate of a mobile phase is 1.0mL/min, the ELSD evaporation temperature is 40-60 ℃, the atomizing gas is nitrogen, and the gas pressure is 350-420 KPa.
2. The method for rapidly extracting and detecting sucralose in food according to claim 1, wherein the method comprises the following steps: the protein and/or fat containing sample, the sauce and sauce product, vinegar, soy sauce, beverage sample are solid samples, semi-solid samples or liquid samples; the mass-volume ratio of the solid sample to the semi-solid sample to the ultrapure water is 1-2 g: 10-20 mL, wherein the mass-to-volume ratio of the liquid sample to the ultrapure water is 2-5 g: 8-5 mL.
3. The method for rapidly extracting and detecting sucralose in food according to claim 1, wherein the method comprises the following steps: in the step (1), the volume ratio of the sample to be extracted to the acetonitrile is 1: 1 to 2.5.
4. The method for rapidly extracting and detecting sucralose in food according to claim 1, wherein the method comprises the following steps: the volume ratio of the total volume of the zinc acetate solution and the potassium ferrocyanide solution in the step (1) to the sample to be extracted is 1-2: 10-20, wherein the volume ratio of the zinc acetate solution to the potassium ferrocyanide solution is 1: 1, wherein the mass concentration of the zinc acetate solution is 200g/L, and the mass concentration of the potassium ferrocyanide solution is 100 g/L.
5. The method for rapidly extracting and detecting sucralose in food according to claim 1, wherein the method comprises the following steps: the mass-volume ratio of the sodium chloride to the sample to be extracted in the step (1) is 3-5 g: 10-20 mL, wherein the mass-volume ratio of the anhydrous magnesium sulfate to the sample to be extracted is 1-2 g: 10-20 mL, wherein the mass ratio of the sodium chloride to the anhydrous magnesium sulfate is 1-3: 2 to 5.
6. The method for rapidly extracting and detecting sucralose in food according to claim 1, wherein the method comprises the following steps: the step (1) of repeatedly extracting the sample to be extracted at the lower layer with acetonitrile refers to repeatedly extracting the sample to be extracted at the lower layer with acetonitrile for two times or more.
7. The method for rapidly extracting and detecting sucralose in food according to claim 1, wherein the method comprises the following steps: combining acetonitrile layers in the step (1), carrying out rotary evaporation to dryness under the conditions of water bath at 46-50 ℃ and negative pressure, dissolving with ultrapure water to obtain 1-4 mL of a dissolved sample, and adding n-hexane, wherein the volume ratio of the n-hexane to the dissolved sample is 1-5: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810228640.6A CN108445118B (en) | 2018-03-20 | 2018-03-20 | Method for rapidly extracting and detecting sucralose in food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810228640.6A CN108445118B (en) | 2018-03-20 | 2018-03-20 | Method for rapidly extracting and detecting sucralose in food |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108445118A CN108445118A (en) | 2018-08-24 |
| CN108445118B true CN108445118B (en) | 2021-07-27 |
Family
ID=63195948
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810228640.6A Active CN108445118B (en) | 2018-03-20 | 2018-03-20 | Method for rapidly extracting and detecting sucralose in food |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108445118B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109580844A (en) * | 2019-01-22 | 2019-04-05 | 梧州市食品药品检验所 | Method that is a kind of while measuring benzoic acid, sorbic acid and saccharin sodium in high grease cake |
| CN109580847B (en) * | 2019-01-22 | 2021-06-01 | 梧州市食品药品检验所 | Method for determining acesulfame in cakes |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101634648B (en) * | 2009-08-21 | 2012-06-27 | 江南大学 | Method for detecting six trace sweetening agents in white spirit by ultra-high pressure liquid chromatography and time-of-flight mass spectrometry |
| CN105372355A (en) * | 2014-08-19 | 2016-03-02 | 内蒙古蒙牛乳业（集团）股份有限公司 | Pre-treatment method for detecting aflatoxin in modulated milk, and detection method using pre-treatment method |
| CN104316611A (en) * | 2014-10-24 | 2015-01-28 | 西安莹朴生物科技股份有限公司 | Method for measuring content of sucralose |
| CN104880516A (en) * | 2015-04-02 | 2015-09-02 | 贵阳学院 | Method for simultaneously detecting six artificial sweeteners in maotai-flavour liquor |
| CN106324150B (en) * | 2016-11-25 | 2019-05-14 | 四川省食品药品检验检测院 | The method of eight kinds of sweeteners in high performance liquid chromatography low-temperature evaporation light scattering determining white wine |
-
2018
- 2018-03-20 CN CN201810228640.6A patent/CN108445118B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN108445118A (en) | 2018-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rezaee et al. | A simple and rapid new dispersive liquid–liquid microextraction based on solidification of floating organic drop combined with inductively coupled plasma-optical emission spectrometry for preconcentration and determination of aluminium in water samples | |
| Dong et al. | Solid phase extraction with high polarity Carb/PSA as composite fillers prior to UPLC-MS/MS to determine six bisphenols and alkylphenols in trace level hotpot seasoning | |
| Chriswell et al. | Chromatographic determination of phenols in water | |
| Barfi et al. | Simplified miniaturized ultrasound-assisted matrix solid phase dispersion extraction and high performance liquid chromatographic determination of seven flavonoids in citrus fruit juice and human fluid samples: hesperetin and naringenin as biomarkers | |
| Campillo et al. | Dispersive liquid–liquid microextraction for the determination of macrocyclic lactones in milk by liquid chromatography with diode array detection and atmospheric pressure chemical ionization ion-trap tandem mass spectrometry | |
| CN108445118B (en) | Method for rapidly extracting and detecting sucralose in food | |
| CN115684442B (en) | Sample pretreatment method and detection method of phenol disinfection byproducts | |
| Hu et al. | Development of a pH-dependent homogeneous liquid-liquid extraction by cold-induced phase separation in acetonitrile/water mixtures for determination of quinolone residues in animal-derived foods | |
| Huang et al. | Optimized QuEChERS method combined with UHPLC-MS/MS for the simultaneous determination of 15 mycotoxins in liquorice | |
| CN108426962B (en) | Method for simultaneously detecting 7 typical mycotoxins in fruits and vegetables | |
| Kong et al. | Chitosan-based adsorption and freeze deproteinization: Improved extraction and purification of synthetic colorants from protein-rich food samples | |
| CN109633028B (en) | Method for detecting spice additive in formula food with special medical application | |
| Farhadi et al. | Dispersive Liquid‐Liquid Microextraction followed by HPLC‐DAD as an efficient and sensitive technique for the determination of patulin from apple juice and concentrate samples | |
| Mahmoudpour et al. | Dispersive liquid–liquid microextraction for HPLC-UV determination of PAHs in milk | |
| Zhang et al. | Determination of chloramphenicol residues in aquatic products using immunoaffinity column cleanup and high performance liquid chromatography with ultraviolet detection | |
| Oishi et al. | Simultaneous LC-MS/MS determination of 18 plant toxins in beverages | |
| Nguyen et al. | Simultaneous determination of 11 water-soluble dyes in food products and beverages by high performance liquid chromatography | |
| CN113325119A (en) | Pesticide residue sample pretreatment concentration method | |
| Oguri et al. | Selective analysis of histamine in food by means of solid-phase extraction cleanup and chromatographic separation | |
| CN103217498A (en) | Method for detecting dicyandiamide in milk powder with LC-MS (liquid chromatography/mass spectrometry) and sample preparation method | |
| CN114669280B (en) | Purifying filler for sulfonamide residue and pretreatment method | |
| Al-Kharosi et al. | A simple method for simultaneous determination of commonly used artificial food colors and preservatives in soda, jam, and yogurt by HPLC-PDA | |
| CN111474279B (en) | Method and kit for detecting macrolide antibiotic compounds | |
| CN101639467A (en) | Method for determining amount of residual florfenicol in aquatic product by using high-efficiency liquid chromatogram fluorescence method | |
| CN105974020B (en) | A kind of method of naphthol yellow S and rhodamine B in LC MS/MS measure fruit juice, beverage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |