CN111289674A - Detection method for simultaneously determining five selenium forms in selenium-rich fruits - Google Patents
Detection method for simultaneously determining five selenium forms in selenium-rich fruits Download PDFInfo
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- 239000011669 selenium Substances 0.000 title claims abstract description 92
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 74
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 64
- 238000001514 detection method Methods 0.000 title claims abstract description 23
- 125000003748 selenium group Chemical group *[Se]* 0.000 title claims abstract description 19
- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 17
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 63
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 27
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- 238000001402 ion-exchange chromatography-inductively coupled plasma mass spectrometry Methods 0.000 claims abstract description 4
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- 150000003343 selenium compounds Chemical class 0.000 claims description 16
- 239000012224 working solution Substances 0.000 claims description 16
- FDKWRPBBCBCIGA-REOHCLBHSA-N (2r)-2-azaniumyl-3-$l^{1}-selanylpropanoate Chemical compound [Se]C[C@H](N)C(O)=O FDKWRPBBCBCIGA-REOHCLBHSA-N 0.000 claims description 14
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- 239000011159 matrix material Substances 0.000 claims description 14
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 230000014759 maintenance of location Effects 0.000 claims description 11
- 229940082569 selenite Drugs 0.000 claims description 10
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 claims description 10
- 238000004364 calculation method Methods 0.000 claims description 8
- 238000004255 ion exchange chromatography Methods 0.000 claims description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 8
- 229960002718 selenomethionine Drugs 0.000 claims description 8
- 125000003130 L-selenocysteinyl group Chemical group O=C([*])[C@@](N([H])[H])([H])C([H])([H])[Se][H] 0.000 claims description 7
- JULROCUWKLNBSN-UHFFFAOYSA-N seleno-DL-cystine Natural products OC(=O)C(N)C[Se][Se]CC(N)C(O)=O JULROCUWKLNBSN-UHFFFAOYSA-N 0.000 claims description 7
- 238000009616 inductively coupled plasma Methods 0.000 claims description 6
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- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 4
- 238000005349 anion exchange Methods 0.000 claims description 4
- 239000012159 carrier gas Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 238000007872 degassing Methods 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
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- 238000000034 method Methods 0.000 abstract description 8
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- BWKOZPVPARTQIV-UHFFFAOYSA-N azanium;hydron;2-hydroxypropane-1,2,3-tricarboxylate Chemical compound [NH4+].OC(=O)CC(O)(C(O)=O)CC([O-])=O BWKOZPVPARTQIV-UHFFFAOYSA-N 0.000 abstract description 2
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- MCAHWIHFGHIESP-UHFFFAOYSA-N selenous acid Chemical compound O[Se](O)=O MCAHWIHFGHIESP-UHFFFAOYSA-N 0.000 description 6
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 5
- 241000220225 Malus Species 0.000 description 4
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
- G01N30/68—Flame ionisation detectors
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- 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
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- G01N2030/065—Preparation using different phases to separate parts of sample
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Abstract
A detection method for simultaneously determining five selenium forms in selenium-rich fruits belongs to the field of foods. Firstly, freeze-drying a sample at the temperature of below 40 ℃ below zero to fully break the wall of plant cells, keeping the selenium form in fruits unchanged, and fully extracting the different forms of selenium; secondly, establishing an extraction method for extracting for 4 hours by using 0.2 percent of citric acid as an extracting agent and oscillating in a water bath at 50 ℃; and finally, establishing a detection method for simultaneously determining 5 selenium forms in the selenium-enriched fruits by using 0.1% citric acid-ammonia water as a mobile phase system (pH is 5) and by using an ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS). The invention has important significance for the source of selenium element in selenium-rich fruits and the enrichment, conversion and migration in the fruits, and the current national standard has no detection technical standard aiming at specific selenium form. In addition, the method has the characteristics of small reagent dosage, simple operation, high extraction rate, short analysis time, low detection limit, good stability, accurate detection and the like, and solves and makes up the defects of the existing detection method.
Description
Technical Field
The invention belongs to the field of foods, and relates to a detection method for detecting five kinds of selenium in selenium-rich fruits.
Background
At present, the national standard for detecting selenium mainly comprises GB 5009.93-2017 'determination of selenium in food', and the method is used for determining total selenium in food, and cannot analyze the form of the selenium, so that the form distribution of the selenium in selenium-enriched fruits cannot be correctly understood and evaluated, and the effect on human health cannot be realized.
The invention establishes a method for extracting citric acid (2g/L) as an extraction reagent and a constant temperature water area oscillation method as an extraction method, and a sample solution is separated by Ion Chromatography (IC) and then is measured by inductively coupled plasma mass spectrometry (ICP-MS).
Disclosure of Invention
The selenium form is difficult to extract due to the low selenium content in the fruit. Aiming at the problems in the prior art, the invention provides a detection method for simultaneously detecting five selenium forms in selenium-rich fruits, solves the technical problems of extraction and detection of the selenium forms in the selenium-rich fruits, improves the detection limit of the method by nearly 10 times, and greatly improves the extraction rate.
In order to achieve the purpose, the invention adopts the following detection steps:
a detection method for simultaneously determining five selenium forms in selenium-rich fruits comprises the following steps:
(1) preparing solution
① 2g/L citric acid solution, weighing 2g citric acid, and diluting to 1000mL with water;
② 1g/L citric acid solution 1g of citric acid was weighed and diluted to 1000mL with water.
(2) Preparing standard solution
① five selenium standards, seleno-amino acid standard with purity of 99%, including selenocysteine [ SeCys2], selenocysteine [ SeCys ], selenomethionine [ SeMet ], selenious acid standard solution [ Se (IV) ], and selenous acid standard solution [ Se (VI) ], wherein the concentration of selenous acid standard solution is 1000 mg/L;
② mixing standard intermediate solution, namely accurately weighing appropriate amount of selenite standard solution [ Se (IV) ], selenate standard solution [ Se (VI) ], and seleno-amino acid standard, and preparing into selenium mixed standard stock solution (calculated by selenium) with concentration of 1 mg/L;
③ matrix matching mixing standard working solution, accurately sucking mixing standard intermediate solution 0.25mL, 0.5mL, 1mL, 2mL, 4mL respectively in 50mL volumetric flask, diluting with 1g/L citric acid solution to scale, shaking up to obtain series matrix matching standard working solution, wherein the concentration of selenium standard solution is 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 μ g/L in sequence.
(3) Sample pretreatment method (preparation of sample solution)
① preprocessing the sample, cleaning the selenium-rich fruit sample, pulverizing, freeze drying at-40 deg.C, and grinding into powder.
② sample solution is prepared by weighing 0.5g dried sample, adding 20mL citric acid (2g/L) into a graduated centrifuge tube, mixing, placing in 50 deg.C constant temperature water bath, shaking for extraction for 4h, taking out, diluting to 25mL, centrifuging at 10000r/min for 10min, collecting supernatant 5mL, adding n-hexane 1mL, mixing by vortex, collecting water phase sample, and passing through 0.22 μm water film.
(4) Ion chromatography-inductively coupled plasma mass spectrometry reference conditions
① ion chromatography separation conditions
Mobile phase: weighing 1.0g of citric acid, dissolving in 1000mL of water, adjusting pH to 5.0 with ammonia water, adding 10mL of methanol, and ultrasonically degassing in a water bath for 10min for later use. A chromatographic column: anion exchange column Hamilton PRP X100(250 mm. times.4 mm). Flow rate: 1.0 mL/min. Sample introduction volume: 50 μ L.
② inductively coupled plasma mass spectrometer conditions
Radio frequency power: 1550W; sampling depth: 9 mm; the collection mass number: se 78; flow rate of carrier gas: 0.7L/min; flow rate of auxiliary gas: 0.48L/min; integration time: 0.5 s.
(5) Qualitative analysis
And (3) determining the matrix matching mixed standard working solution prepared in the step (2) and the sample solution prepared in the step (3) according to the conditions of an ion chromatography-inductively coupled plasma mass spectrometer, recording the chromatographic retention time of each compound in the standard working solution and the sample solution, and determining that the corresponding compound is detected in the sample when the chromatogram consistent with the chromatographic retention time of five selenium in the standard solution is detected in the sample solution. Part of the chromatographic retention time of the sample may have small shifts, so that it is difficult to determine which selenium compound is by retention time, in this case, the selenium compound can be determined on-machine by adding the selenium compound standard solution into the sample, and if the unknown chromatographic peak of the sample is coincident with the chromatographic peak of the known selenium compound standard solution, the selenium compound can be determined.
(6) Quantitative determination
①, the matrix matching mixed standard working solution is respectively measured according to the reference conditions of the instrument to obtain the chromatographic peak area of the corresponding standard solution, the retention time T/min is used as the abscissa, the response chromatographic peak area I/cps is used as the ordinate, a standard curve is drawn, and the content of each selenium form is calculated through the standard curve according to the corresponding peak area in the chromatogram of the sample solution.
The content of the target object in the sample solution is quantified by using a standard curve external standard method, and the calculation formula is as follows:
in the formula: x represents the content of each selenium compound in the sample, mg/kg;
c, concentration of each selenium compound in the sample solution, namely mu g/L;
v, the volume of the sample solution is determined to be mL;
m is sample weighing mass, g;
k is the dilution multiple.
The calculation results retain three significant digits.
② the linear range of the five kinds of selenium is 5-100 mug/L, the detection limits of selenocysteine [ SeCys2], selenocysteine [ SeCys ], selenite [ Se (IV) ], selenomethionine [ SeMet ], selenate [ Se (VI) ], are respectively 0.5 mug/L, 0.8 mug/L, 0.6 mug/L, 1.2 mug/L and 0.6 mug/L.
The invention has the beneficial effects that:
(1) the analysis and detection are carried out by combining Ion Chromatography (IC) with inductively coupled plasma mass spectrometry (ICP-MS), the selenium in five selenium forms can be qualitatively and quantitatively analyzed within 15 minutes, and the analysis time is shorter than that of the existing method;
(2) the freeze drying technology is adopted for pretreatment, so that the cell walls of plants can be fully damaged, the extraction efficiency is improved, the migration and transformation of the form of selenium can be prevented, the detection limit is improved by nearly 10 times, and the problem that the content of selenium in fruits is too low and difficult to detect is solved;
(3) the selenium form can be ensured to be stable by adopting a citric acid-ammonia water buffer salt mobile phase system, and the citric acid (2g/L) is adopted to extract in a 50 ℃ water bath oscillation system, so that the extraction rate can be improved to the maximum extent on the premise of ensuring the selenium form to be stable, and the problems of easy structure fracture and low recovery rate under the secondary ultrasonic extraction of selenomethionine SeMet are solved;
(4) the method has the characteristics of small reagent dosage, simple operation, high extraction rate, low detection limit, good stability, accurate detection and the like, solves and makes up the defects of the existing detection method, and meets the requirement of trace element morphological analysis.
Drawings
FIG. 1 is a chromatogram of five selenium species, in which selenocysteine [ SeCys2], selenocysteine [ SeCys ], selenite [ Se (IV) ], selenomethionine [ SeMet ], selenate [ Se (VI) ], are present in the chromatogram from left to right;
FIG. 2 is a standard curve of selenocysteine [ SeCys2], the concentrations of which are 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L and 80 μ g/L in sequence;
FIG. 3 is a standard curve of Selenocysteine [ SeCys ], the concentrations of which are 5 mug/L, 10 mug/L, 20 mug/L, 40 mug/L and 80 mug/L in sequence;
FIG. 4 is a selenite [ Se (IV) ] standard curve, with concentrations of 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 μ g/L in sequence;
FIG. 5 is a selenomethionine [ SeMet ] standard curve with concentrations of 5. mu.g/L, 10. mu.g/L, 20. mu.g/L, 40. mu.g/L, and 80. mu.g/L in sequence;
FIG. 6 is a selenate [ Se (VI) ] standard curve, the concentrations of which are 5 mug/L, 10 mug/L, 20 mug/L, 40 mug/L and 80 mug/L in sequence;
FIG. 7 is a chromatogram of a blank apple sample without five selenium;
FIG. 8 is a chromatogram of a selenium enriched apple sample.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1 determination of five selenium forms in selenium-enriched apples
(1) Sample pretreatment
① preprocessing the sample, cleaning the selenium-rich apple sample, pulverizing, freeze-drying at-40 deg.C, and grinding into powder.
② sample solution is prepared by weighing 0.5g dried sample, adding 20mL citric acid (2g/L) into a graduated centrifuge tube, mixing, placing in 50 deg.C constant temperature water bath, shaking for extraction for 4h, taking out, diluting to 25mL, centrifuging at 10000r/min for 10min, collecting supernatant 5mL, adding n-hexane 1mL, mixing by vortex, collecting water phase sample, and passing through 0.22 μm water film.
(2) Reference conditions for ion chromatography-tandem mass spectrometry
① ion chromatography separation conditions
Mobile phase: weighing 1.0g of citric acid, dissolving in 1000mL of water, adjusting pH to 5.0 with ammonia water, adding 10mL of methanol, and ultrasonically degassing in a water bath for 10min for later use. A chromatographic column: anion exchange column Hamilton PRP X100(250 mm. times.4 mm). Flow rate: 1.0 mL/min. Sample introduction volume: 50 μ L.
② inductively coupled plasma mass spectrometer conditions
Radio frequency power: 1550W; sampling depth: 9 mm; the collection mass number: 78 (Se); flow rate of carrier gas: 0.7L/min; flow rate of auxiliary gas: 0.48L/min; integration time: 0.5 s.
(3) Preparation of Standard solutions
① five selenium standards, seleno-amino acid standard with purity of 99%, including selenocysteine [ SeCys2], selenocysteine [ SeCys ], selenomethionine [ SeMet ], selenious acid standard solution [ Se (IV) ], and selenous acid standard solution [ Se (VI) ], wherein the concentration of selenous acid standard solution is 1000 mg/L;
② mixing standard intermediate solution, namely accurately weighing appropriate amount of selenite standard solution [ Se (IV) ], selenate standard solution [ Se (VI) ], and seleno-amino acid standard, and preparing into selenium mixed standard stock solution (calculated by selenium) with concentration of 1 mg/L;
③ matrix matching mixing standard working solution, accurately sucking mixing standard intermediate solution 0.25mL, 0.5mL, 1mL, 2mL, 4mL respectively in 50mL volumetric flask, diluting with 1g/L citric acid solution to scale, shaking up to obtain series matrix matching standard working solution, wherein the concentration of selenium standard solution is 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 μ g/L in sequence.
(4) Quantitative determination
And respectively measuring the matrix matching mixed standard working solution according to the reference conditions of the instrument to obtain the chromatographic peak area of the corresponding standard solution, drawing a standard curve by taking the retention time T/min as a horizontal coordinate and the response chromatographic peak area I/cps as a vertical coordinate, and calculating the content of each selenium form by taking the corresponding peak area in the chromatogram of the sample solution through the standard curve.
The content of the target object in the sample solution is quantified by using a standard curve external standard method, and the calculation formula is as follows:
in the formula: x represents the content of each selenium compound in the sample, mg/kg;
c, concentration of each selenium compound in the sample solution, namely mu g/L;
v, the volume of the sample solution is determined to be mL;
m is sample weighing mass, g;
k is the dilution multiple.
The calculation results retain three significant digits.
(5) Recovery and repeatability of spiked samples
The spiking recovery test was performed by adding three concentration levels of 5. mu.g/L, 20. mu.g/L, and 50. mu.g/L to a control blank (sample not rich in selenium) (n-3), respectively.
TABLE 2 spiking recovery experiment
Tab2 Addition recovery test
Table 2 shows that the average recovery rate of the three standard addition levels is above 82%, the relative standard deviation is less than 7%, and experimental data show that the recovery rate and the repeatability of the method in the example meet the requirements of the detection method.
Example 2 determination of inorganic and organic selenium in selenium-rich cherries
(1) Sample pretreatment
① preprocessing the sample, cleaning the selenium-rich cherry sample, pulverizing, freeze-drying at-40 deg.C, and grinding into powder.
② sample solution is prepared by weighing 0.5g dried sample, adding 20mL citric acid (2g/L) into a graduated centrifuge tube, mixing, placing in 50 deg.C constant temperature water bath, shaking for extraction for 4h, taking out, diluting to 25mL, centrifuging at 10000r/min for 10min, collecting supernatant 5mL, adding n-hexane 1mL, mixing by vortex, collecting water phase sample, and passing through 0.22 μm water film.
(2) Reference conditions for ion chromatography-tandem mass spectrometry
① ion chromatography separation conditions
Mobile phase: weighing 1.0g of citric acid, dissolving in 1000mL of water, adjusting pH to 5.0 with ammonia water, adding 10mL of methanol, and ultrasonically degassing in a water bath for 10min for later use. A chromatographic column: anion exchange column Hamilton PRP X100(250 mm. times.4 mm). Flow rate: 1.0 mL/min. Sample introduction volume: 50 μ L.
② inductively coupled plasma mass spectrometer conditions
Radio frequency power: 1550W; sampling depth: 9 mm; the collection mass number: 78 (Se); flow rate of carrier gas: 0.7L/min; flow rate of auxiliary gas: 0.48L/min; integration time: 0.5 s.
(3) Preparation of Standard solutions
① selenium standard substance comprising selenite standard solution [ Se (IV) ]and selenate standard solution [ Se (VI) ]with concentration of 1000 mg/L;
② mixing standard intermediate solution, accurately weighing appropriate amount of selenite standard solution [ Se (IV) ] and selenate standard solution [ Se (VI) ] to obtain selenium mixed standard stock solution (calculated as selenium) with concentration of 1 mg/L;
③ matrix matching mixing standard working solution, accurately sucking mixing standard intermediate solution 0.25mL, 0.5mL, 1mL, 2mL, 4mL respectively in 50mL volumetric flask, diluting with 1g/L citric acid solution to scale, shaking up, and using as series matrix matching standard working solution with concentration of 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 μ g/L in sequence.
(4) Quantitative determination of inorganic selenium (selenite [ Se (IV) ], selenate [ Se (VI) ])
And respectively measuring the matrix matching mixed standard working solution according to the reference conditions of the instrument to obtain the chromatographic peak area of the corresponding standard solution, drawing a standard curve by taking the retention time T/min as a horizontal coordinate and the response chromatographic peak area I/cps as a vertical coordinate, and calculating the content of each selenium form by using the corresponding peak area in the chromatogram of the sample solution through the standard curve.
The content of the target object in the sample solution is quantified by using a standard curve external standard method, and the calculation formula is as follows:
in the formula: x represents the content of each selenium compound in the sample, mg/kg;
c, concentration of each selenium compound in the sample solution, namely mu g/L;
v, the volume of the sample solution is determined to be mL;
m is sample weighing mass, g;
k is the dilution multiple.
The calculation results retained three significant figures and the sample was found to have an inorganic selenium content (on a dry basis) of 0.122 mg/kg.
(5) Determination of total selenium: weighing 0.3g of freeze-dried cherry sample, placing the sample in a digestion tank, adding 5mL of nitric acid and 2mL of hydrogen peroxide, and carrying out microwave digestion. After digestion, removing acid on an electric hot plate to 1mL, transferring to a 50mL volumetric flask, and directly measuring by using inductively coupled plasma mass spectrometry to obtain the cherry sample total selenium (calculated on a dry basis) content of 0.367 mg/kg.
(6) The content of organic selenium: the content of the organic selenium is the total selenium content minus the inorganic selenium content, and 0.367mg/kg-0.122mg/kg is 0.245mg/kg, so that the content of the organic selenium (calculated by a dry basis) in the cherry is measured to be 0.245mg/kg, and the cherry organic selenium accounts for 66.8 percent of the total selenium.
The above-mentioned embodiments only express the embodiments of the present invention, but not should be understood as the limitation of the scope of the invention patent, it should be noted that, for those skilled in the art, many variations and modifications can be made without departing from the concept of the present invention, and these all fall into the protection scope of the present invention.
Claims (1)
1. A detection method for simultaneously determining five selenium forms in selenium-rich fruits is characterized by comprising the following steps:
(1) preparing solution
① 2g/L citric acid solution;
② 1g/L citric acid solution;
(2) preparing standard solution
① five selenium standards, seleno-amino acid standard with purity of 99%, including selenocysteine [ SeCys2], selenocysteine [ SeCys ], selenomethionine [ SeMet ], selenious acid radical standard solution [ Se (IV) ], and selenous acid radical standard solution [ Se (VI) ], wherein the concentration of the selenous acid radical standard solution is 1000 mg/L;
② mixing standard intermediate solution, namely accurately weighing appropriate amount of selenite standard solution [ Se (IV) ], selenate standard solution [ Se (VI) ], and seleno-amino acid standard, and preparing into selenium mixed standard stock solution with selenium concentration of 1 mg/L;
③ matrix matching mixing standard working solution, which is to accurately absorb 0.25mL, 0.5mL, 1mL, 2mL and 4mL of the mixing standard intermediate solution into a 50mL volumetric flask, dilute the solution to a scale with 1g/L citric acid solution, shake the solution uniformly to serve as a series of matrix matching standard working solutions, wherein the concentrations of the selenium standard solution are 5 mug/L, 10 mug/L, 20 mug/L, 40 mug/L and 80 mug/L in sequence;
(3) sample pretreatment method
① preprocessing the sample, cleaning the selenium-rich fruit sample, pulverizing, freeze drying at-40 deg.C, and grinding into powder;
② sample solution is prepared by weighing 0.5g dried sample in a centrifuge tube, adding 2g/L citric acid, mixing, placing in 50 deg.C constant temperature water bath, shaking for extraction for 4 hr, centrifuging at 10000r/min for 10min, collecting supernatant 5mL, adding n-hexane 1mL, mixing by vortex, collecting water phase sample, and passing through 0.22 μm water film;
(4) ion chromatography-inductively coupled plasma mass spectrometry reference conditions
① ion chromatography separation conditions
Mobile phase: weighing 1.0g of citric acid, dissolving in 1000mL of water, adjusting the pH value to 5.0 with ammonia water, adding 10mL of methanol, and ultrasonically degassing in a water bath for 10min for later use; a chromatographic column: anion exchange column Hamilton PRP X100(250 mm. times.4 mm); flow rate: 1.0 mL/min; sample introduction volume: 50 mu L of the solution;
② inductively coupled plasma mass spectrometer conditions
Radio frequency power: 1550W; sampling depth: 9 mm; the collection mass number: se 78; flow rate of carrier gas: 0.7L/min; flow rate of auxiliary gas: 0.48L/min; integration time: 0.5 s;
(5) qualitative analysis
Determining the matrix matching mixed standard working solution prepared in the step (2) and the sample solution prepared in the step (3) according to the conditions of an ion chromatography-inductively coupled plasma mass spectrometer, recording the chromatographic retention time of each compound in the standard working solution and the sample solution, and determining that a corresponding compound is detected in the sample when a chromatogram consistent with the chromatographic retention time of five selenium in the standard solution is detected in the sample solution; when the retention time of part of the sample chromatogram shifts, the selenium compound standard solution is added into the sample for on-machine determination, and if the unknown chromatographic peak of the sample is superposed with the chromatographic peak of the known selenium compound standard solution, the sample is identified as the selenium compound;
(6) quantitative determination
①, respectively measuring the matrix matching mixed standard working solution according to the reference conditions of the instrument to obtain the chromatographic peak area of the corresponding standard solution, drawing a standard curve by taking the retention time T/min as the abscissa and the response chromatographic peak area I/cps as the ordinate, and calculating the content of each selenium form by the corresponding peak area in the chromatogram of the sample solution through the standard curve;
the content of the target object in the sample solution is quantified by using a standard curve external standard method, and the calculation formula is as follows:
in the formula: x represents the content of each selenium compound in the sample, mg/kg;
c, concentration of each selenium compound in the sample solution, namely mu g/L;
v, the volume of the sample solution is determined to be mL;
m is sample weighing mass, g;
k is the dilution multiple;
the calculation result retains three significant digits;
② the linear range of the five kinds of selenium is 5-100 mug/L, the detection limits of selenocysteine [ SeCys2], selenocysteine [ SeCys ], selenite [ Se (IV) ], selenomethionine [ SeMet ], selenate [ Se (VI) ], are respectively 0.5 mug/L, 0.8 mug/L, 0.6 mug/L, 1.2 mug/L and 0.6 mug/L.
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