CN114705767A - Analysis and detection method for selenium form in rice - Google Patents

Abstract

The invention relates to the technical field of food component analysis, in particular to an analysis and detection method for selenium form in rice. The method comprises the following steps: taking rice, performing total selenium digestion sample preparation on the rice, and if the rice subjected to total selenium digestion sample preparation is selenium-rich rice, crushing the rice and preparing the sample to obtain a rice sample; carrying out enzymolysis treatment on the rice sample by using an extracting solution for enzymolysis to obtain a rice selenium morphological analysis sample solution; and performing qualitative and quantitative analysis on the rice selenium form analysis sample liquid by adopting a high performance liquid chromatography-inductively coupled plasma tandem mass spectrometry instrument to analyze the selenium form in the rice. The invention aims to solve the problem that the complete separation and online measurement of the selenium form in the rice are difficult to realize due to low sensitivity and accuracy when the conventional analysis and detection method is used for measuring the selenium form in the rice.

Description

Analysis and detection method for selenium form in rice

Technical Field

The invention relates to the technical field of food component analysis, in particular to a method for analyzing and detecting the form of selenium in rice.

Background

Selenium is one of the trace elements essential for human life activity, and the organic selenium mainly includes macromolecular selenium such as selenoprotein, seleno-nucleic acid and selenoglycopolysaccharide, and small molecular selenides such as selenocysteine, selenomethionine, methyl selenocysteine and selenoethylthionine. At present, organic selenium in rice, such as selenoprotein, seleno amino acids, selenium polypeptide and selenium polysaccharide, is converted into physiologically active substances in human bodies and is absorbed and utilized by the human bodies.

At present, methods for detecting selenium in rice include Atomic Fluorescence Spectrometry (AFS), Atomic Absorption Spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS) and the like, in the national standard of food selenium determination, an ICP-MS method is added to GB 5009.93-2017 'determination of selenium in food safety national standard food' on the basis of the old national standard as a third method, and an ICP-MS method is adopted in a first method of GB 5009.268-2016 'determination of multiple elements in food safety national standard food', so that compared with two spectrophotometry methods and inductively coupled plasma emission spectrometry (ICP-OES), the method has the advantages of rapidness, sensitivity, low reagent toxicity and the like.

However, according to the difference of the above detection technologies, the digested sample of organic selenium in rice can be directly used for measuring total selenium, and the total selenium content-inorganic selenium content ═ organic selenium content is mostly used, the measuring process is complicated, only the total organic selenium content can be obtained, and the specific organic selenium content in different forms cannot be directly obtained.

Meanwhile, in the above conventional detection method, the first ionization energy of selenium is high (I1 ═ 9.75eV), so that the ionization efficiency is relatively low, and there are interferences of various polyatomic ions, such as⁴⁰Ar³⁶Ar⁺To pair⁷⁶Se、⁴⁰Ar³⁸Ar⁺To pair⁷⁸Se、⁴⁰Ar³⁷Ar⁺To pair⁷⁷Se、³⁷Cl³⁷Cl⁺To pair⁷⁴Se and⁸¹Br¹h pair⁸²Se and the like, and the interference of the polyatomic ions reduces the sensitivity and the accuracy of the ICP-MS technology on selenium determination, so that the complete separation and online determination of the selenium form in the rice cannot be realized.

Disclosure of Invention

Aiming at the defects in the prior art, the invention solves the problem that the complete separation and online measurement of the selenium form in the rice are difficult to realize due to low sensitivity and accuracy when the conventional analysis and detection method is used for measuring the selenium form in the rice.

In order to achieve the purpose, the invention adopts the following technical scheme: a method for analyzing and detecting the form of selenium in rice comprises the following steps:

taking rice, performing total selenium digestion sample preparation on the rice, and crushing the rice to prepare a sample to obtain a rice sample if the rice subjected to total selenium digestion sample preparation is selenium-rich rice;

carrying out enzymolysis treatment on the rice sample by using an extracting solution for enzymolysis to obtain a rice selenium morphological analysis sample solution;

and qualitatively and quantitatively analyzing the rice selenium form analysis sample liquid by adopting a high performance liquid chromatography-inductively coupled plasma tandem mass spectrometry combination instrument to analyze the selenium form in the rice.

Preferably, the total selenium digestion sample preparation method adopts one of a microwave digestion method, a wet digestion method or a high-pressure digestion method.

Preferably, the extract is prepared by mixing pure water, trypsin, proteinase K and pronase.

Preferably, the components in the extracting solution are as follows by mass:

pure water: 22-28 parts; trypsin: 8-12 parts; and (3) protease K: 8-12 parts; 8-12 parts of pronase.

Preferably, the enzymatic hydrolysis treatment comprises the following specific steps: adding the extracting solution into the rice sample, and carrying out water bath oscillation for 4-6 h at the temperature of 40-65 ℃.

Preferably, the high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer adopts an oxygenation mode Se (m/z 80- >96), a chromatographic column is a Hamilton PRP-X100 analytical column, and a mobile phase is 5mmol/L citric acid solution with the pH value of 5.0.

Preferably, the specific parameter values used by the high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer are as follows:

radio frequency power is 1550W, radio frequency voltage is 1.30V, sampling depth is 8.0-10.0mm, carrier gas flow rate is 0.77-1.07L/min, oxygen proportion is 10% -30%, helium flow rate is 2-5L/min, collision mode is O₂The detection element is⁷⁸Se and⁸⁰se, mass transfer of⁷⁸Se¹⁶O(m/z 78->94)，⁸⁰Se¹⁶O (m/z 80->96)。

Preferably, the time for the qualitative and quantitative analysis is 15 to 20 minutes.

Preferably, the specific operation method of the high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer comprises the following steps:

separating the rice selenium morphological analysis sample liquid by HPLC liquid phase, atomizing in an inductively coupled plasma tandem mass spectrometer atomizer, and ionizing the atomized sample liquid into plasma by high pressure;

the plasma enters a first-stage four-stage rod Q1 through a taper hole to carry out first-step mass screening, the screened plasma enters a collision pool, oxygen m/z is introduced into the collision pool to be 16, and collision reaction is carried out;

and the ions after the collision reaction are subjected to a second-step mass screening through a second-stage four-stage rod Q2, so that mass transfer is performed, and a plasma meeting the requirements is screened out.

Compared with the prior art, the beneficial effect that this scheme produced is:

the rice selenium form analysis sample liquid is obtained after the rice sample is subjected to enzymolysis through the extracting solution, qualitative and quantitative analysis is carried out on the rice selenium form analysis sample liquid by adopting a high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer, complete separation and online determination of the selenium form in the rice can be realized in one-time sample introduction, and the requirement of rapidly and accurately detecting SeCys in the rice can be met₂Content of MeSeCys, Se (IV), SeMet and Se (VI)。

Drawings

In order to more clearly illustrate the embodiments of the present invention, the drawings, which are required to be used in the embodiments, will be briefly described below. In all the drawings, the elements or parts are not necessarily drawn to actual scale.

FIG. 1 is a table showing a microwave digestion method used in a method for analyzing and detecting the form of selenium in rice according to the present invention;

FIG. 2 is a table of the preparation of 5 types of standard solutions of mixed forms of selenium analyzed and detected in rice by the method of example 2;

FIG. 3 is a table of the preparation of standard solutions of mixed selenium forms in the analytical test of rice by the method of example 2;

FIG. 4 is a table showing the detection limits and precision of SeMet-selenomethionine detected by rice analysis by the method of example 2;

FIG. 5 shows SeCys detected by rice analysis according to example 2₂-a table of detection limits and precision of selenocysteine;

FIG. 6 is a table showing the detection limits and precision of Me-SeCys-methylselenocysteine detected by rice analysis according to example 2;

FIG. 7 is a table showing the detection limit and precision of Se (IV) -selenite in rice analyzed by the method of example 2;

FIG. 8 is a table showing the detection limit and precision of Se (VI) -selenate detected from rice by the method of example 2;

FIG. 9 is an ion flow graph of the complete time frame of 5 forms of selenium detected by analysis of rice by the method of example 2;

FIG. 10 is a graph of retention times over a complete time range for 5 forms of selenium detected from rice analyzed by the method of example 2;

FIG. 11 shows SeCys detected by rice analysis according to example 2₂Singly calibrating a bitmap;

FIG. 12 is a MeSeCys single bitmap detected from rice analysis by the method of example 2;

FIG. 13 is a single bitmap of Se (IV) detected from rice analyzed by the method of example 2;

FIG. 14 is a bitmap of a single set of SeMet detected by rice analysis according to the method of example 2;

FIG. 15 is a Se (VI) single-scale bitmap detected by rice analysis according to the method in example 2;

FIG. 16 is a flow chart of the operation of the HPLC-ICP-MS in the method for analyzing and detecting the form of selenium in rice according to the present invention;

FIG. 17 is a comparison graph of the detection effect of different modes of the HPLC-ICP-MS used in the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

Example 1

A method for analyzing and detecting the form of selenium in rice comprises the following steps:

step one, taking rice, carrying out total selenium digestion sample preparation on the rice, and if the rice subjected to total selenium digestion sample preparation is selenium-rich rice, crushing the rice and preparing the sample to obtain a rice sample.

In this step, referring to table 1, the weight of the selected rice may be 0.5 g; meanwhile, digesting and sample preparation are carried out on the rice by adopting a microwave digestion method, and 50mL of rice sample liquid after digestion and sample preparation is obtained. Therefore, whether the selected rice is selenium-enriched rice or not can be confirmed through the rice sample liquid. Wherein, the microwave digestion method can be replaced by a wet digestion method or a high-pressure digestion method.

And step two, carrying out enzymolysis treatment on the rice sample by using the extracting solution for enzymolysis to obtain a rice selenium morphological analysis sample solution.

In this step, the extract is prepared by mixing pure water, trypsin, proteinase K and pronase.

Wherein the extracting solution comprises the following components in parts by weight:

pure water: 22 parts of (A); trypsin: 12 parts of (1); and (3) protease K: 8 parts of a mixture; and 12 parts of chain protease. Namely, the specific components in the extracting solution are pure water: 22mL, trypsin: 12 mg; and (3) protease K: 8 mg; pronase 12 mg.

The enzymolysis treatment comprises the following specific steps: adding the extractive solution into rice sample, and oscillating in water bath at 40 deg.C for 6 hr.

And thirdly, qualitatively and quantitatively analyzing the rice selenium form analysis sample liquid by adopting a high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer to analyze the selenium form in the rice.

In this step, referring to fig. 9, the hplc-icp tandem mass spectrometer is specifically an agilent 8900 icp tandem mass spectrometer.

Meanwhile, the high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer adopts an oxygenation mode Se (m/z 80- >96), a chromatographic column is a Hamilton PRP-X100 analytical column, and a mobile phase is 5mmol/L citric acid solution with the pH value of 5.0.

The specific parameter values used by the high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer are as follows:

radio frequency power is 1550W, radio frequency voltage is 1.30V, sampling depth is 8.0-10.0mm, carrier gas flow rate is 0.77-1.07L/min, oxygen proportion is 10% -30%, helium flow rate is 2-5L/min, collision mode is O₂The detection element is⁷⁸Se and⁸⁰se, mass transfer of⁷⁸Se¹⁶O(m/z 78->94)，⁸⁰Se¹⁶O (m/z 80->96)。

Referring to fig. 8, the specific operation method of the hplc-icp tandem mass spectrometer includes:

the rice selenium form analysis sample liquid is subjected to HPLC liquid phase separation, enters an inductively coupled plasma tandem mass spectrometer atomizer for atomization, the atomized sample liquid is ionized into plasma through high pressure, the plasma enters a first-stage four-stage rod Q1 through a taper hole to be subjected to first-step mass screening m/z (78), the screened mass number ions enter a collision pool, oxygen m/z (16) is introduced into the collision pool to perform collision reaction, and the rice selenium form analysis sample liquid is not screened, and then is directly screened out.

Meanwhile, ions after the collision reaction are subjected to a second-step mass screening m/z 94 through a second-stage four-stage rod Q2, mass transfer is carried out, ions with inconsistent mass numbers are screened out, and interference is eliminated; meanwhile, the mass number ions that are not screened out do not enter the Q2 system. Thereby obtaining a highly sensitive plasma. The detected count value (CPS) and concentration value are more sensitive by adopting an oxygenation mode through a high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer.

Meanwhile, the time for qualitative and quantitative analysis using a high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer (HPLC-ICP-MS/MS) was 15 minutes.

Example 2

Referring to fig. 1 to 17, on the basis of embodiment 1, the differences between this embodiment and embodiment 1 are as follows:

in the second step, the components in the extracting solution are as follows according to the parts by weight:

pure water: 22 parts of (A); trypsin: 12 parts of (1); and (3) protease K: 8 parts of a mixture; and 12 parts of chain protease. Namely, the specific components in the extracting solution are pure water: 25mL, trypsin: 10 mg; and (3) protease K: 10 mg; 10mg of pronase. The rice can be subjected to enzymolysis more quickly by using the extracting solution.

The enzymolysis treatment comprises the following specific steps: adding the extractive solution into rice sample, and oscillating in water bath at 60 deg.C for 4.5 h.

And step five, performing qualitative and quantitative analysis by using a high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer for 18 minutes.

Example 3

On the basis of embodiment 1, the present embodiment differs from embodiment 1 as follows:

in the second step, the components in the extracting solution are as follows according to the parts by mass:

pure water: 28 parts of (1); trypsin: 8 parts; and (3) protease K: 12 parts of (a); 8 parts of chain protease. Namely, the specific components in the extracting solution are pure water: 28mL, trypsin: 8 mg; and (3) protease K: 12 mg; 8mg of pronase. The rice can be subjected to enzymolysis more quickly by using the extracting solution.

The enzymolysis treatment comprises the following specific steps: adding the extractive solution into rice sample, and oscillating in water bath at 65 deg.C for 4 hr.

In the fifth step, the specific operation method of the high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer comprises the following steps:

the rice selenium form analysis sample liquid is subjected to HPLC liquid phase separation, enters an inductively coupled plasma tandem mass spectrometer atomizer for atomization, the atomized sample liquid is ionized into plasma through high pressure, the plasma enters a first-stage four-stage rod Q1 through a taper hole to be subjected to first-step mass screening m/z (80), the screened mass number ions enter a collision pool, oxygen m/z (16) is introduced into the collision pool to perform collision reaction, and the ions are not screened and are directly screened out.

Meanwhile, ions after the collision reaction are subjected to a second-step mass screening m/z (96) through a second-stage four-stage rod Q2, mass transfer is carried out, ions with inconsistent mass numbers are screened out, and interference is eliminated; meanwhile, the mass number ions that are not screened out do not enter the Q2 system. Thereby obtaining a highly sensitive plasma.

The time for qualitative and quantitative analysis using HPLC-ICP-MS was 20 min.

Through the above embodiments 1 to 3, the rice selenium form analysis sample liquid is obtained by performing enzymolysis on the rice sample with the extracting solution, and then qualitative and quantitative analysis is performed on the rice selenium form analysis sample liquid with a high performance liquid chromatography-inductively coupled plasma tandem mass spectrometry (HPLC-ICP-MS/MS), so that complete separation and online determination of the selenium form in 5 of rice can be realized in one sample injection, and rapid and accurate detection of the selenium form in 5 of rice including sec cys can be satisfied₂Content of MeSeCys, Se (IV), SeMet, and Se (VI).

The rice was analyzed and tested by the above analysis and test method in example 2, and the conclusion is as follows:

the results are shown in FIGS. 1 to 8, which give SeCys₂The detection limits of MeSeCys, Se (IV), SeMet and Se (VI) are respectively (0.002, 0.004, 0.005, 0.002, 0.001) mg/kg. Curve range: 0-20 mug/L.

From fig. 9 to fig. 17, it can be seen that the correlation coefficient R of the linear range obtained by the method is between 0.9996 and 1.000, the linear range is good, and the precision is high. Thereby realizing the complete separation and the on-line determination in one sample introduction, and meeting the requirement of quickly and accurately detecting SeCys₂Content of MeSeCys, Se (IV), SeMet, and Se (VI).

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims (9)

1. The method for analyzing and detecting the form of selenium in rice is characterized by comprising the following steps of:

taking rice, performing total selenium digestion sample preparation on the rice, and if the rice subjected to total selenium digestion sample preparation is selenium-rich rice, crushing the rice and preparing the sample to obtain a rice sample;

carrying out enzymolysis treatment on the rice sample by using an extracting solution for enzymolysis to obtain a rice selenium morphological analysis sample solution;

and performing qualitative and quantitative analysis on the rice selenium form analysis sample liquid by adopting a high performance liquid chromatography-inductively coupled plasma tandem mass spectrometry instrument to analyze the selenium form in the rice.

2. The method for analyzing and detecting the form of selenium in rice according to claim 1, wherein the total selenium digestion sample preparation method adopts one of a microwave digestion method, a wet digestion method or a high pressure digestion method.

3. The method of claim 2, wherein the extract is prepared by mixing purified water, trypsin, proteinase K and pronase.

4. The method for analyzing and detecting the form of selenium in rice as claimed in claim 3, wherein the extract comprises the following components in parts by weight:

pure water: 22-28 parts; trypsin: 8-12 parts; and (3) protease K: 8-12 parts; 8-12 parts of pronase.

5. The method for analyzing and detecting the form of selenium in rice according to claim 1 or 4, wherein the enzymatic hydrolysis treatment comprises the following specific steps:

adding the extracting solution into the rice sample, and carrying out water bath oscillation for 4-6 h at the temperature of 40-65 ℃.

6. The method for analyzing and detecting the form of selenium in rice according to claim 5, wherein the high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer adopts an oxygenation mode Se (m/z 80- >96), the chromatographic column is a Hamilton PRP-X100 analytical column, and the mobile phase is 5mmol/L citric acid solution with pH of 5.0.

7. The method for analyzing and detecting the form of selenium in rice according to claim 1, wherein specific parameter values used by the high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer are as follows:

radio frequency power of 1550W, radio frequency voltage of 1.30V, sampling depth of 8.0-10.0mm, carrier gas flow rate of 0.77-1.07L/min, oxygen proportion of 10-30%, helium flow rate of 2-5L/min, and collision mode of O₂The detection element is⁷⁸Se and⁸⁰se, mass transfer of⁷⁸Se¹⁶O（m/z 78->94），⁸⁰Se¹⁶O（m/z 80->96）。

8. The method of claim 7, wherein the qualitative and quantitative analysis is performed for a time period of 15 to 20 minutes.

9. The method for analyzing and detecting the form of selenium in rice according to claim 7, wherein the specific operation method of the high performance liquid chromatography-inductively coupled plasma tandem mass spectrometer comprises the following steps:

separating the rice selenium morphological analysis sample liquid by HPLC liquid phase, atomizing in an inductively coupled plasma tandem mass spectrometer atomizer, and ionizing the atomized sample liquid into plasma by high pressure;

the plasma enters a first-stage four-stage rod Q1 through a taper hole to carry out first-step mass screening, the screened plasma enters a collision pool, and oxygen m/z =16 is introduced into the collision pool to carry out collision reaction;

and the ions after the collision reaction are subjected to a second-step mass screening through a second-stage four-stage rod Q2, so that mass transfer is performed, and a plasma meeting the requirements is screened out.

Images