CN111707746A - Method for detecting different arsenic form contents in food - Google Patents
Method for detecting different arsenic form contents in food Download PDFInfo
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- CN111707746A CN111707746A CN202010545927.9A CN202010545927A CN111707746A CN 111707746 A CN111707746 A CN 111707746A CN 202010545927 A CN202010545927 A CN 202010545927A CN 111707746 A CN111707746 A CN 111707746A
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- 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
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- 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
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- 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
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- 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
- G01N2030/042—Standards
- G01N2030/047—Standards external
Abstract
The invention discloses a method for detecting different arsenic form contents in food, which comprises the following steps: preparing a mixed standard solution; making a standard curve; sample pretreatment: soaking a sample in 0.10-0.3mol/L nitric acid solution overnight, hot-leaching in a thermostat at 80-100 ℃ for 1-3h, taking out, cooling to room temperature, centrifuging at 4000r/min for 30min, filtering supernatant with 0.45 mu m organic filter membrane, and performing sample injection determination; sample detection: and (3) sucking 50-500 mu L of the sample solution, injecting the sample solution into a liquid chromatogram-atomic fluorescence spectrum combined instrument to obtain a chromatogram, performing qualitative determination by using retention time, and obtaining the content of each component in the sample solution according to a standard curve. The method for detecting the contents of different arsenic forms in the food is a method for detecting four arsenic forms in rice, health care products and wine samples based on the HPLC-AFS combined technology, has the characteristics of good accuracy, high precision, high sensitivity, good linear correlation and the like, saves time and labor, and can greatly improve the experimental efficiency.
Description
Technical Field
The invention relates to a detection method based on HPLC-AFS combination, in particular to a detection method for different arsenic form contents in food.
Background
According to the specification of liquid chromatography-atomic fluorescence spectrometry in national standard GB5009.11-2014 (determination of total arsenic and inorganic arsenic in food safety national standard), inorganic arsenic in food is extracted by dilute nitric acid, then is separated by liquid chromatography, and a separated target compound reacts with KBH4 in an acidic environment to generate a gaseous arsenic compound which is then determined by an atomic fluorescence spectrometer. Qualitative according to retention time, and quantitative according to external standard method. According to the method, when the sampling amount is 1g and the constant volume is 20ml, the detection limit of inorganic arsenic in the rice is 0.02 mg/kg.
In the current national standard, only two inorganic arsenicals (trivalent arsenic AsIII and pentavalent arsenic AsV) are analyzed, the detection limit of the two inorganic arsenicals is not established respectively after the two inorganic arsenicals are used for 12min, and the qualitative and quantitative analysis of MMA and DMA of the methyl arsenicals is not carried out.
However, DMA and MMA are 2B carcinogens published by the world health organization international cancer research institution, are ubiquitous in the natural world, are easily absorbed by plants and animals, influence biological metabolism and enzyme activity, and have great harm to human beings and ecological environment.
Therefore, a method for detecting the content of different arsenic forms in foods with various arsenic forms is needed.
Disclosure of Invention
The invention aims to provide a method for detecting different arsenic form contents in food, which shortens the analysis time to 4min, greatly improves the detection efficiency, saves a large amount of manpower and material resources, and can be used for detecting trace arsenic forms.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for detecting different arsenic morphological contents in food comprises the following steps:
preparing a mixed standard solution: preparing the mixed standard solution by using deionized water, and enabling the concentration of various arsenic forms in the mixed standard solution to be 100 mu g/L;
and (3) preparing a standard curve: taking a mixed standard solution with a corresponding volume, and adding a proper amount of deionized water to ensure that the concentration of various arsenic forms is 1-100 mu g/L; drawing a standard curve according to the signal intensity corresponding to different arsenic form concentrations;
sample pretreatment: soaking a sample in 0.10-0.3mol/L nitric acid solution overnight, hot-leaching in a thermostat at 80-100 ℃ for 1-3h, taking out, cooling to room temperature, centrifuging at 4000r/min for 30min, filtering supernatant with 0.45 mu m organic filter membrane, and performing sample injection determination;
sample detection: and (3) sucking 50-500 mu L of the sample solution, injecting the sample solution into a liquid chromatogram-atomic fluorescence spectrum combined instrument to obtain a chromatogram, performing qualitative determination by using retention time, and obtaining the content of each component in the sample solution according to a standard curve.
Optionally, the various arsenic forms include trivalent arsenic AsIII, pentavalent arsenic AsV, monomethylarsenate MMA, and dimethylarsenate DMA.
Optionally, the sample solution is subjected to multiple measurements, and the average value of the multiple measurements is taken as the content of different forms of arsenic in the sample solution.
Alternatively, the conditions used for liquid chromatography are as follows: a chromatographic column: c18 or equivalent columns; mobile phase: 2-10mmol/L tetrabutylammonium hydroxide, 2-10mmol/L malonic acid, 2-10mmol/L ammonia water, 2-10% methanol, and pH of 2-10; and (3) an elution mode: isocratic elution; flow rate: 0.5-1.5 mL/min; sample introduction volume: 50-500 μ L.
Optionally, the atomic fluorescence detection conditions are as follows: negative high pressure: 285V; main current/auxiliary current: 40/40 mA; liquid carrying: 2-30% hydrochloric acid solution; reducing agent: 5-50g/L potassium borohydride solution; rotation speed of peristaltic pump: 35 RPM; flow rate of carrier gas: 400 mL/min; shielding the airflow rate: 800 mL/min.
Optionally, the glassware is soaked in a nitric acid solution (1+4) for 24h, repeatedly washed with water, and finally washed clean with deionized water.
Optionally, the mobile phase is filtered with 0.45 μm water system filter membrane, and degassed with helium or ultrasound for 20min before use.
Optionally, the detection method is used for detecting trivalent arsenic AsIII, pentavalent arsenic AsV, monomethylarsenic acid MMA and dimethyl arsenic acid DMA in rice, health products or wine samples.
The invention has the following beneficial effects: the method for detecting the contents of different arsenic forms in the food is a method for detecting four arsenic forms in rice, health care products and wine samples based on the HPLC-AFS combined technology, has the characteristics of good accuracy, high precision, high sensitivity, good linear correlation and the like, saves time and labor, and can greatly improve the experimental efficiency.
Drawings
FIG. 1 shows the spectrum of AsIII, DMA, MMA and AsV (10. mu.g/L) standard solutions according to the present invention.
Detailed Description
The technical solution of the present invention is further described below with reference to the following embodiments and the accompanying drawings.
Example 1
The embodiment provides a method for detecting different arsenic form contents in food, which comprises the following steps:
s10 preparation of mixed standard solution
In this embodiment, the mixed standard solution is prepared by using deionized water, and the concentrations of various arsenic forms in the mixed standard solution are all 100 μ g/L; wherein the various arsenic forms include trivalent arsenic AsIII, pentavalent arsenic AsV, monomethylarsenate MMA and dimethylarsenate DMA.
S20, preparation of Standard Curve
Taking a mixed standard solution with a corresponding volume, and adding a proper amount of deionized water to ensure that the concentration of various arsenic forms is 1-100 mu g/L; preferably, the concentration of each arsenic form can be 2. mu.g/L, 5. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L, 50. mu.g/L, 100. mu.g/L, and a standard curve is drawn according to the signal intensity corresponding to the concentration of different arsenic forms, and the standard curve is shown in FIG. 1.
S30 sample pretreatment
Soaking a sample in 0.10-0.3mol/L nitric acid solution overnight, hot-leaching for 1-3h in a thermostat at 80-100 ℃, taking out and cooling to room temperature, centrifuging for 30min at 4000r/min, taking supernatant, filtering by a 0.45-micron organic filter membrane, performing sample determination, and performing a reagent blank experiment.
S40, detecting the sample
And (3) sucking 50-500 mu L of the sample solution, injecting the sample solution into a liquid chromatogram-atomic fluorescence spectrum combined instrument to obtain a chromatogram, performing qualitative determination by using retention time, and obtaining the content of different arsenic forms in the sample solution according to a standard curve.
Preferably, the sample solution is measured a plurality of times, and the average of the plurality of measurements is taken as the content of different forms of arsenic in the sample solution.
Wherein, the conditions used in the liquid chromatography are as follows: a chromatographic column: c18 or equivalent columns; mobile phase: 2-10mmol/L tetrabutylammonium hydroxide, 2-10mmol/L malonic acid, 2-10mmol/L ammonia water, 2-10% methanol, and pH of 2-10; and (3) an elution mode: isocratic elution; flow rate: 0.5-1.5 mL/min; sample introduction volume: 50-500 μ L.
Preferably, the mobile phase is degassed before use using a 0.45 μm aqueous membrane filter, helium or sonication for 20 min.
Wherein, the conditions used for atomic fluorescence detection are as follows: negative high pressure: 285V; main current/auxiliary current: 40/40 mA; liquid carrying: 2-30% hydrochloric acid solution; reducing agent: 5-50g/L potassium borohydride solution; rotation speed of peristaltic pump: 35 RPM; flow rate of carrier gas: 400 mL/min; shielding the airflow rate: 800 mL/min.
In the detection process of S40, all glassware needs to be soaked in nitric acid solution (1+4) for 24h, repeatedly washed by water, and finally washed clean by deionized water.
In the method for detecting the content of different arsenic forms in the food of the embodiment, when the addition recovery rate test is performed, the mixed standard solution is added into the blank sample, so that the concentration of each form of arsenic reaches 5 μ g/L and 10 μ g/L, and the result of the standard addition recovery test shows that the standard addition recovery rate of different concentrations of four arsenic forms reaches 80-120%.
In addition, according to the method for detecting the content of different forms of arsenic in the food, 10 mug/L of mixed standard solution is continuously tested for 7 times under the repetitive condition, and the precision of the concentration of each form of arsenic is calculated to be less than 3%, which shows that the method is high in stability and has practical application value
According to the specification of 6.3.4 minimum detection quantity in JJG 1151-2018 liquid chromatogram-atomic fluorescence combined instrument verification procedure, the mixed standard solution with the concentration of 3 mu g/L is injected, and the peak height value of the signal of each component is compared with the noise in the injection state, so that the minimum detection quantity of each component is obtained. Calculating to obtain a method detection limit: when the sampling amount is 0.5g and the constant volume is 10mL, the detection limit is as follows: AsIII: 0.004 mg/kg; DMA: 0.0052 mg/kg; MMA: 0.0056 mg/kg; AsV: 0.015mg/kg, which is far lower than the detection limit of the prior art, and has the characteristic of high sensitivity.
Moreover, various arsenic forms show good linear relation in the concentration range of 2-100 mug/L, and the correlation coefficient is more than 0.9990.
The method for detecting the contents of different arsenic forms in the food is based on the HPLC-AFS combined technology, has the characteristics of good accuracy, high precision, high sensitivity, good linear correlation and the like, saves time and labor, and can greatly improve the experimental efficiency.
Example 2
The embodiment provides a method for detecting contents of different arsenic forms in food, which adopts a HPLC-AFS combined technology to determine the contents of the different arsenic forms.
Linear range: and (3) taking 5 10ml volumetric flasks, accurately adding 0.20ml, 0.50ml, 1.00ml, 1.50ml and 2.00ml of 100 mu g/L mixed standard solution respectively, and diluting with deionized water to the scale. The concentrations of the standard series of solutions at this time were: 2. mu.g/L, 5. mu.g/L, 10. mu.g/L, 15. mu.g/L, 20. mu.g/L.
And (5) absorbing 100 mu L of standard series solution, injecting the standard series solution into a liquid chromatogram-atomic fluorescence spectrum combined instrument for analysis to obtain a chromatogram, and determining the chromatogram by retention time. And (4) drawing a standard curve by taking the concentration of the target compound in the standard series solution as a horizontal coordinate and the chromatographic peak area as a vertical coordinate. Obtaining a working curve, wherein R values all meet more than 0.9990. The standard solution chromatogram is shown in FIG. 1.
AsIII：IF＝108.0066*C-32.1361 R＝0.9994；
DMA：IF＝109.1887*C-36.8463 R＝0.9994；
MMA：IF＝100.7194*C+6.3527 R＝0.9996；
AsV：IF＝66.7653*C-61.2637 R＝0.9993。
Accuracy: samples in the application range of the embodiment are selected, the samples are labeled according to various arsenic morphological concentrations of 5 mug/L and 10 mug/L, and the recovery rate data are obtained after blank samples are deducted.
TABLE 1 Standard recovery experiment for 5. mu.g/L and 10. mu.g/L arsenic form of different samples
According to experimental results, the standard recovery rates of the four arsenic forms with different concentrations reach 80-120%.
Precision: the mixed standard solution of 10 mug/L is continuously tested for 7 times, and the precision of each component is calculated as follows: 2.5%, 2.7%, 2.5%, 2.6%.
Detection limit: injecting a mixed standard solution with the concentration of 3 mug/L, comparing the peak height value of a signal of each component with noise in the injection state, and calculating to obtain the minimum detection quantity of each component as follows: AsIII: 0.020 ng; DMA: 0.026 ng; MMA: 0.028 ng; AsV: 0.075 ng.
The method comprises the following steps: when the sampling amount is 0.5g and the constant volume is 10mL, the detection limits of various arsenic forms are respectively as follows: AsIII: 0.004 mg/kg; DMA: 0.0052 mg/kg; MMA: 0.0056 mg/kg; AsV: 0.015 mg/kg.
Example 3
The embodiment provides a method for detecting contents of different arsenic forms in food, which adopts a HPLC-AFS combined technology to determine the contents of the different arsenic forms.
Sample pretreatment: care should be taken not to contaminate the sample during sampling and preparation. The rice and the health care products are crushed evenly and put into a clean polyethylene bottle for sealing and storage for later use. The alcohol beverage was sealed in a clean polyethylene bottle and kept in a refrigerator at 4 ℃ for later use.
Sample extraction: approximately 0.5g of rice or a nutraceutical (exactly 0.001g) was weighed into a 50mL plastic centrifuge tube, and 20mL of a 0.15mol/L nitric acid solution was added and left overnight. Hot-leaching at 90 deg.C for 2 hr, and shaking for 1min every 0.5 hr. After extraction, taking out and cooling to room temperature, and centrifuging for 30min at 4000 r/min. Taking supernatant, filtering with 0.45 μm organic filter membrane, and sampling for determination. A blank test was conducted in the same manner.
Or sucking 1mL of alcoholic liquor sample by using a pipette gun, adding 20mL of 0.15mol/L nitric acid solution into a 50mL plastic centrifuge tube, and standing overnight. Hot-leaching at 90 deg.C for 2 hr, and shaking for 1min every 0.5 hr. After extraction, taking out and cooling to room temperature, and centrifuging for 30min at 4000 r/min. Taking supernatant, filtering with 0.45 μm organic filter membrane, and sampling for determination. A blank test was conducted in the same manner.
Measurement of sample solution: and (3) sucking 100 mu L of sample solution, injecting the sample solution into a liquid chromatogram-atomic fluorescence spectrum combined instrument to obtain a chromatogram, and determining the chromatogram by retention time. And obtaining the content of each component in the sample solution according to the standard curve, and carrying out parallel measurement for no less than two times.
The detection results are as follows:
TABLE 2 detection results of arsenic in various forms in different samples
| Sample name | Sample weighing | Volume of constant volume | AsIII | DMA | MMA | AsV |
| Selenium-rich rice | 0.5g | 10ml | 17.37μg/kg | 17.29μg/kg | — | — |
| Selenium supplementing tablet | 0.5g | 10ml | — | — | — | 33.15μg/kg |
| Two years of red daughter | 1ml | 10ml | 7.78μg/L | 10.61μg/L | — | — |
| Yellow rice wine | 1ml | 10ml | 5.45μg/L | 8.54μg/L | — | — |
The detection method for the different arsenic morphological contents in the food is based on the liquid chromatography-atomic fluorescence spectrometry combined technology, is suitable for separating and measuring trivalent arsenic AsIII, pentavalent arsenic AsV, monomethylarsenic acid MMA and dimethyl arsenic acid DMA in rice, health products and wine samples, and is only used for 4 min; and the standard curve, the standard recovery rate, the precision and the detection limit are verified by experiments, so that the requirements of chemical analysis are met.
The sequence of the above embodiments is only for convenience of description and does not represent the advantages and disadvantages of the embodiments.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (8)
1. A method for detecting the content of different arsenic forms in food is characterized by comprising the following steps:
preparing a mixed standard solution: preparing the mixed standard solution by using deionized water, and enabling the concentration of various arsenic forms in the mixed standard solution to be 100 mu g/L;
and (3) preparing a standard curve: taking a mixed standard solution with a corresponding volume, and adding a proper amount of deionized water to ensure that the concentration of various arsenic forms is 1-100 mu g/L; drawing a standard curve according to the signal intensity corresponding to different arsenic form concentrations;
sample pretreatment: soaking a sample in 0.10-0.3mol/L nitric acid solution overnight, hot-leaching in a thermostat at 80-100 ℃ for 1-3h, taking out, cooling to room temperature, centrifuging at 4000r/min for 30min, filtering supernatant with 0.45 mu m organic filter membrane, and performing sample injection determination;
sample detection: and (3) sucking 50-500 mu L of the sample solution, injecting the sample solution into a liquid chromatogram-atomic fluorescence spectrum combined instrument to obtain a chromatogram, performing qualitative determination by using retention time, and obtaining the content of each component in the sample solution according to a standard curve.
2. The method for detecting the content of different arsenic forms in food according to claim 1, wherein the arsenic forms comprise trivalent arsenic AsIII, pentavalent arsenic AsV, monomethylarsenic acid MMA and dimethylarsenic acid DMA.
3. The method according to claim 2, wherein the sample solution is measured a plurality of times, and an average value of the plurality of measurements is used as the content of different forms of arsenic in the sample solution.
4. The method for detecting the morphological content of different arsenic in food according to claim 3, wherein the conditions of the liquid chromatography are as follows: a chromatographic column: c18 or equivalent columns; mobile phase: 2-10mmol/L tetrabutylammonium hydroxide, 2-10mmol/L malonic acid, 2-10mmol/L ammonia water, 2-10% methanol, and pH of 2-10; and (3) an elution mode: isocratic elution; flow rate: 0.5-1.5 mL/min; sample introduction volume: 50-500 μ L.
5. The method for detecting the morphological content of different arsenic in food according to claim 3, wherein the atomic fluorescence detection is performed under the following conditions: negative high pressure: 285V; main current/auxiliary current: 40/40 mA; liquid carrying: 2-30% hydrochloric acid solution; reducing agent: 5-50g/L potassium borohydride solution; rotation speed of peristaltic pump: 35 RPM; flow rate of carrier gas: 400 mL/min; shielding the airflow rate: 800 mL/min.
6. The method for detecting the morphological content of different arsenic in food as claimed in claim 1, wherein the glassware is soaked in nitric acid solution (1+4) for 24h, washed repeatedly with water and finally washed clean with deionized water.
7. The method for detecting the morphological content of different arsenic in food as claimed in claim 4, wherein the mobile phase is degassed by 0.45 μm water system filter membrane, helium or ultrasound for 20min before use.
8. The method for detecting the morphological content of different arsenic in the food according to claim 1, wherein the method is used for detecting trivalent arsenic AsIII, pentavalent arsenic AsV, monomethylarsenic acid MMA and dimethyl arsenic acid DMA in rice, health products or wine samples.
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