Selenium form standard quality control sample in nut matrix as well as preparation method and application of selenium form standard quality control sample

Abstract

The invention provides a selenium form standard quality control sample in a nut matrix as well as a preparation method and application thereof, and belongs to the technical field of standard substances. The method comprises the following steps: performing primary crushing on seed kernels of Brazil nuts to obtain seed kernel particles; sequentially performing degreasing treatment and secondary crushing on the seed kernel particles to obtain degreased powder; spraying a selenite solution on the degreased powder to obtain composite powder; the composite powder is sequentially subjected to freeze drying, third crushing and mixing, and the selenium form standard quality control sample in the nut matrix is obtained. The standard quality control sample for the selenium form in the nut matrix, prepared by the method disclosed by the invention, has good uniformity and stability, and by adopting the standard quality control sample, the accuracy and the reliability of a content detection result of the selenium form (selenite and / or selenomethionine) in plant products such as nuts can be improved.

Description

Technical Field

[0001] This invention relates to the field of standard material technology, and in particular to a standard quality control sample of selenium speciation in nut matrix, its preparation method and application. Background Technology

[0002] Selenium, as one of the essential trace elements for the human body, possesses physiological functions such as anti-oxidation, anti-aging, tumor inhibition, and cardiovascular protection. With increasing public awareness of selenium nutrition, selenium-enriched foods (such as selenium-enriched rice, walnuts, and vegetables) are gaining widespread popularity in the consumer market. However, the forms of selenium in food vary significantly, and different forms of selenium have different effects on human health. Studies have shown that inorganic selenium (such as sodium selenite and sodium selenate) has high toxicity, while organic selenium (including selenomethionine, selenocysteine, and selenocysteine) not only has lower toxicity but also higher bioavailability. It is worth noting that both inorganic and organic selenium can be effectively absorbed and utilized by the human body within appropriate concentration ranges, but the safe intake threshold for inorganic selenium is significantly lower than that for organic selenium.

[0003] Currently, selenium speciation detection technology has reached a relatively mature stage. Conventional detection procedures typically involve first separating the target analyte using liquid-phase separation technology, followed by quantitative determination using atomic fluorescence spectrometry (AFS) or inductively coupled plasma mass spectrometry (ICP-MS). However, due to the scarcity of effective standard materials, the reliability of selenium speciation detection results can currently only be indirectly verified through spiked recovery rates. But recovery rates only reflect the accuracy of the detection process (such as instrument measurement), and cannot effectively evaluate the extraction efficiency of the sample during pretreatment (i.e., the degree to which the target selenium speciation is effectively released from and retained in the sample matrix). This lack of a quality control system can easily lead to deviations in detection results, potentially posing a risk to the quality control of the selenium-enriched product market.

[0004] Therefore, there is an urgent need to develop a standard quality control sample for selenium speciation in plant matrices, such as nut matrices, that has a simple preparation process and accurate determination results, so as to effectively verify the accuracy and reliability of selenium speciation detection methods. Summary of the Invention

[0005] The purpose of this invention is to provide a standard quality control sample for selenium speciation in nut matrix, its preparation method, and its application. The standard quality control sample for selenium speciation in nut matrix prepared by the method of this invention has good uniformity and stability. Using this standard quality control sample helps to improve the accuracy and reliability of the detection results of selenium speciation (selenite and / or selenomethionine) content in plant products such as nuts.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a standard quality control sample of selenium speciation in a nut matrix, comprising the following steps: The kernel of the abalone fruit is first crushed to obtain kernel particles; The kernel particles are sequentially degreased and then pulverized to obtain degreased powder. A selenite solution was sprayed onto the degreased powder to obtain a composite powder; The composite powder was freeze-dried, then pulverized and mixed in sequence to obtain the standard quality control sample of selenium speciation in the nut matrix.

[0007] Preferably, the degreasing agent used in the degreasing treatment includes petroleum ether, and the ratio of the kernel granules to the degreasing agent is 1g:5~8mL; the temperature of the degreasing treatment is 15~30℃; the degreasing treatment is carried out under oscillation conditions, the oscillation rate is 150~180rpm, and the oscillation time is 8~12h; the degreasing treatment and the second pulverization further include: allowing the liquid obtained after the degreasing treatment to stand and separating the solid and liquid in sequence, and collecting the solid material; the standing time is 25~35min.

[0008] Preferably, the process after the second crushing further includes: sieving the material obtained after the second crushing, and collecting the undersize material as the degreased powder; the aperture of the sieve used for sieving is 0.250~0.425mm.

[0009] Preferably, the concentration of the selenite solution is 0.2~2 mg / L, and the ratio of the defatted powder to the selenite solution is 1~3 g: 1 mL; The preparation method of the composite powder includes the following steps: laying the defatted powder to form a layer, spraying the selenite solution onto the layer in the form of a spray, and allowing it to stand for absorption to obtain the composite powder; The thickness of the layer is 0.3~0.6mm; the spraying pressure is 0.2~0.4MPa and the height is 40~60cm; the static absorption temperature is 15~30℃ and the time is 1~3h.

[0010] Preferably, the freeze-drying step includes: laying the composite powder to form a layer, and then performing pre-freezing, first freeze-drying and second freeze-drying in sequence; The thickness of the layup is 2-3 cm; the pre-freezing temperature is -40 to -60°C, the pressure is atmospheric pressure, and the time is 2-6 h; the first freeze-drying temperature is -10 to -30°C, the vacuum degree is 5-15 Pa, and the time is 8-16 h; the second freeze-drying temperature is 20-40°C, the vacuum degree is 3-8 Pa, and the time is 3-6 h.

[0011] Preferably, the process between the third crushing and the mixing further includes: sieving the material obtained after the third crushing and collecting the undersize material; the sieve used for sieving has an aperture of 0.075~0.180mm.

[0012] Preferably, the mixing method is three-dimensional rotational mixing, the rotational speed of the three-dimensional rotational mixing is 18~30 rpm, and the time is 6~10 h.

[0013] This invention provides a standard quality control sample of selenium speciation in nut matrix prepared by the preparation method described above.

[0014] This invention provides the application of the standard quality control sample of selenium speciation in nut matrix described above in the detection of selenite content and / or selenomethionine content in plant samples.

[0015] Preferably, the plant-based sample includes a nut sample.

[0016] Beneficial Effects: The selenium speciation standard control sample for nut matrices prepared using the method of this invention exhibits good homogeneity and stability. Using this standard control sample helps improve the accuracy and reliability of selenium speciation (selenite and / or selenomethionine) content detection results in plant-based products such as nuts. Specifically, this invention uses abalone fruit as raw material, which not only has a high content of natural selenium but also a high content of organic selenium such as selenomethionine, while the proportion of other easily degradable organic selenium speciations is extremely low, effectively avoiding interference with the overall stability of the standard control sample. After defatting, the matrix effect of the sample is significantly reduced. Then, selenite is added as an inorganic selenium source, and the final standard control sample contains both stable inorganic selenium (selenite) and organic selenium (selenomethionine). These two selenium speciations are the main forms of selenium in plant-based products such as nuts, effectively meeting the technical requirements for accurate quality control in the detection of such products. Meanwhile, the method of the present invention is simple to operate and does not use heat treatment process during preparation, thereby ensuring the chemical stability of selenite and selenomethionine, so that the standard quality control sample can be stably stored for more than 12 months under low temperature (4℃) conditions. Attached Figure Description

[0017] Figure 1 The chromatogram is for the standard solution; Figure 2 The chromatogram is of the selenium speciation standard control sample prepared in the nut matrix in Example 1. Detailed Implementation

[0018] This invention provides a method for preparing a standard quality control sample of selenium speciation in a nut matrix, comprising the following steps: The kernel of the abalone fruit is first crushed to obtain kernel particles; The kernel particles are sequentially degreased and then pulverized to obtain degreased powder. A selenite solution was sprayed onto the degreased powder to obtain a composite powder; The composite powder was freeze-dried, then pulverized and mixed in sequence to obtain the standard quality control sample of selenium speciation in the nut matrix.

[0019] In this invention, unless otherwise specified, all raw materials used are commercially available products well known to those skilled in the art or prepared using methods well known to those skilled in the art.

[0020] This invention involves first crushing the kernel of abalone fruit to obtain kernel particles. In one embodiment, the abalone fruit can originate from Brazil, specifically from the states of Acre, Amazonas, or Pará. Preferably, the abalone fruit is shelled to obtain the kernel, which is then first crushed. In one embodiment, the kernel particle size can be 1.5~2.5 mm. This invention does not specifically limit the method of first crushing, as long as kernel particles of the above-mentioned particle size are obtained. Specifically, in this embodiment, a pulverizer is used for the first crushing.

[0021] After obtaining the seed kernel granules, the present invention sequentially performs a defatting treatment and a second pulverization on the seed kernel granules to obtain defatted powder. In one embodiment of the present invention, the defatting agent used in the defatting treatment may include petroleum ether; the ratio of seed kernel granules to defatting agent may be 1g:5~8mL, specifically 1g:5mL, 1g:6mL, 1g:7mL, or 1g:8mL; the defatting treatment temperature may be 15~30℃, specifically 15℃, 20℃, 25℃, or 30℃; the defatting treatment may be carried out under oscillation conditions, the oscillation rate may be 150~180rpm, specifically 150rpm, 160rpm, 170rpm, or 180rpm; the oscillation time may be 8~12h, specifically 8h, 9h, 10h, 11h, or 12h. The present invention removes oil from the seed kernel granules through defatting treatment. In this embodiment of the invention, the kernel granules are placed in a conical flask, a degreasing agent is added, and the flask is then sealed with a cap equipped with a small vent. The flask containing the kernel granules and the degreasing agent is then placed in a constant-temperature shaker for degreasing treatment. As one embodiment of the invention, the degreasing treatment preferably further includes: allowing the degreasing material to stand and then separating it into solid and liquid phases, and then collecting the solid material for the second pulverization; the standing time can be 25-35 minutes, specifically 30 minutes, as standing facilitates solid-liquid separation in this embodiment; the solid-liquid separation can be performed by vacuum filtration. Specifically, a pulverizer is used for the second pulverization in this embodiment.

[0022] In one embodiment of the present invention, the second pulverization process preferably further includes: sieving the material obtained after the second pulverization, and collecting the undersize material as the defatted powder; the aperture of the sieve used for sieving can be 0.250~0.425mm, specifically 0.250mm. Using a sieve with the above-mentioned aperture in this embodiment of the present invention helps to ensure sample uniformity; if the sieve aperture is too large, it may lead to insufficient uniformity of the subsequent samples; if the sieve aperture is too small, it may result in too low a yield of undersize material, leading to excessive sample loss.

[0023] After obtaining the defatted powder, the present invention sprays a selenite solution onto the defatted powder to obtain a composite powder. The present invention uses selenite as the inorganic selenium source, which is beneficial for improving sample stability; while if selenate is used, it is prone to reduction reactions in the nut matrix, leading to unstable detection results; furthermore, the main component of inorganic selenium in selenium-enriched products from plant matrices is selenite. In one embodiment of the present invention, the selenite solution is prepared by mixing selenite and water; the selenite can be sodium selenite; and the water can be ultrapure water. In one embodiment of the present invention, the concentration of the selenite solution can be 0.2~2 mg / L, specifically 0.2 mg / L, 0.5 mg / L, 0.8 mg / L, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L or 2 mg / L; the ratio of the defatted powder to the selenite solution can be 1~3 g:1 mL, specifically 1 g:1 mL, 1.5 g:1 mL, 2 g:1 mL, 2.5 g:1 mL or 3 g:1 mL.

[0024] In one embodiment of the present invention, the preparation method of the composite powder may include the following steps: laying the degreased powder to form a layer, spraying the selenite solution onto the layer in a spray form, and allowing it to stand for absorption to obtain the composite powder. In one embodiment of the present invention, the thickness of the layer can be 0.3~0.6 mm, specifically 0.3 mm, 0.4 mm, 0.5 mm, or 0.6 mm; if the layer is too thin, the laying operation becomes significantly more difficult and time-consuming; if the layer is too thick, such as ≥0.8 mm, it will lead to insufficient spraying of the selenite solution, resulting in uneven sample processing. In a specific embodiment of the present invention, the degreased powder is laid on a stainless steel tray to form a layer.

[0025] In one embodiment of the present invention, the spraying pressure can be 0.2~0.4MPa, specifically 0.2MPa, 0.25MPa, 0.3MPa, 0.35MPa, or 0.4MPa; the height can be 40~60cm, specifically 40cm, 45cm, 50cm, 55cm, or 60cm. Specifically, in this embodiment, the selenite solution is placed in an electric sprayer and sprayed onto the layup in a spray form; the spraying time is based on ensuring that the selenite solution is completely sprayed. In this embodiment, the work area is divided into several independent units, with sufficient isolation between units to avoid cross-interference; after each unit completes its work independently, the samples are combined. This method effectively ensures that subsequent spraying operations in the work area will not affect the completed area; the continuous spraying time in each unit during the spraying process can be 2~6s, specifically 2s, 3s, 4s, 5s, or 6s. In this embodiment of the invention, spraying under the above conditions helps to ensure uniform spraying of the selenite solution.

[0026] In one embodiment of the present invention, the temperature for static absorption can be 15~30℃, specifically 15℃, 20℃, 25℃ or 30℃; the time can be 1~3h, specifically 1h, 2h or 3h. Under the above conditions, static absorption allows the target substance to fully contact the absorption medium and achieve an ideal mass transfer equilibrium.

[0027] After obtaining the composite powder, the present invention sequentially freeze-dries, pulverizes, and mixes the composite powder to obtain a standard quality control sample of selenium speciation in the nut matrix. As one embodiment of the present invention, the freeze-drying step may include: laying the composite powder into a layer, and then sequentially pre-freezing, first freeze-drying, and second freeze-drying.

[0028] In one embodiment of the present invention, the thickness of the layup can be 2-3 cm. In another embodiment, the pre-freezing temperature can be -40 to -60°C, specifically -40°C, -45°C, -50°C, -55°C, or -60°C; the pressure can be atmospheric pressure; and the time can be 2-6 hours, specifically 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In another embodiment, the first freeze-drying temperature can be -10 to -30°C, specifically -10°C, -15°C, -20°C, -25°C, or -30°C; the vacuum degree can be 5-15 Pa, specifically 5 Pa, 10 Pa, or 15 Pa; and the time can be 8-16 hours, specifically 8 hours, 10 hours, 12 hours, 14 hours, or 16 hours. In one embodiment of the present invention, the temperature of the second freeze-drying can be 20~40℃, specifically 25℃, 30℃, 35℃ or 40℃; the vacuum degree can be 3~8Pa, specifically 3Pa, 4Pa, 5Pa, 6Pa, 7Pa or 8Pa; and the time can be 3~6h, specifically 3h, 4h, 5h or 6h.

[0029] In this embodiment of the invention, a pulverizer is specifically used for the third pulverization. As one embodiment of the invention, the third pulverization and the mixing process preferably further include: sieving the material obtained after the third pulverization and collecting the undersize material; the aperture of the sieve used for sieving can be 0.075~0.180mm, specifically 0.150mm.

[0030] In one embodiment of the present invention, the mixing method can be three-dimensional rotary mixing. The rotational speed of the three-dimensional rotary mixing can be 18-30 rpm, specifically 18 rpm, 22 rpm, 24 rpm, 26 rpm, or 30 rpm; the time can be 6-10 hours, specifically 6 hours, 8 hours, or 10 hours. Specifically, in this embodiment, a high-efficiency mixer can be used for the three-dimensional rotary mixing. Under the above conditions, the three-dimensional rotary mixing of the present invention can achieve uniform dispersion and sufficient contact of materials in the radial, axial, and tangential dimensions, effectively avoiding problems such as uneven local concentration or mixing dead zones.

[0031] After mixing, the present invention preferably encapsulates the selenium speciation standard quality control sample in the nut matrix in a glass bottle and stores it under cold storage (temperature 0~4℃).

[0032] This invention provides a standard quality control sample of selenium speciation in nut matrix prepared by the preparation method described above.

[0033] This invention provides the application of the standard quality control sample of selenium speciation in nut matrix described above in the detection of selenite content and / or selenomethionine content in plant samples.

[0034] In one embodiment of the present invention, the plant sample may include a nut sample, which may include abalone fruit.

[0035] This invention does not specifically limit the method and conditions for detecting the selenite and / or selenomethionine content in plant samples using the selenium speciation standard control sample from the nut matrix; conventional methods and conditions in the art can be used. Specifically, for the detection of selenium speciation content in plant products, the selenium speciation standard control sample from the nut matrix described in this invention and the sample to be tested are subjected to simultaneous full-process pretreatment and instrumental measurement. When the measurement result of the selenium speciation standard control sample from the nut matrix is ​​within the specified allowable error range, it can be determined that the detection result of the sample to be tested has passed effective quality control, and its measurement data is accurate and reliable.

[0036] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0037] This invention determined the total selenium content and selenium speciation of different types of nuts. Table 1 shows the results of the determination of total selenium content and selenium speciation of different types of nuts. Analysis of Table 1 shows that the total selenium content of Brazilian abalone nuts is significantly higher than that of nuts from other regions, and its selenium speciation is mainly selenomethionine, which has high stability. In contrast, abalone nuts from Peru and Bolivia, although containing only selenomethionine, have relatively low selenium content. Furthermore, the selenium content of other common nuts such as pumpkin seeds, hazelnuts, pine nuts, and sunflower seeds is generally insufficient, making it difficult to meet the requirements for quality control sample raw materials. Sunflower seeds and pumpkin seeds also contain easily degradable selenocysteine. Although some walnut samples from selenium-rich areas can achieve high selenium content levels, meeting the basic requirements for quality control sample preparation, the presence of selenocysteine ​​may affect the long-term stability of the quality control samples. Based on the above analysis, this invention ultimately selected Brazilian abalone nuts as the preferred raw material for preparing standard quality control samples.

[0038] Table 1. Results of determination of total selenium content and selenium speciation in different types of nuts

[0039] Example 1 The preparation method of the standard quality control sample for selenium speciation in nut matrix is ​​as follows: S1. Using abalone fruit (specifically from Acre, Brazil) as raw material, the kernel is obtained after shelling. The kernel is then preliminarily crushed using a pulverizer to obtain kernel particles (particle size 1.5~2.5mm). S2. Place the seed kernel granules in a conical flask, add petroleum ether to the conical flask according to the ratio of seed kernel granules to petroleum ether of 1g:6mL, seal the flask with a cap equipped with a small vent hole, and then place the conical flask containing the seed kernel granules and petroleum ether in a constant temperature shaker for degreasing treatment. The conditions for the degreasing treatment include: temperature of 20℃, shaking speed of 150rpm, shaking time of 8h, standing for 30min after shaking, filtering the obtained liquid to remove oil, and collecting the solid material as degreased material. S3. The degreased material is crushed using a pulverizer and sieved using a sieve with a pore size of 0.250mm. The material passing through the sieve is collected to obtain degreased powder. S4. Selenite (specifically sodium selenite) is mixed with ultrapure water to prepare a selenite solution with a concentration of 0.5 mg / L. The degreasing powder is spread evenly on a stainless steel tray to form a 0.5 mm thick layer. The selenite solution is sprayed evenly onto the degreasing powder using an electric sprayer at a ratio of 3 g: 1 mL (specifically, the working area is divided into several independent units, and each unit is kept sufficiently isolated to avoid cross-interference; after each unit has completed its work independently, the samples are combined). The spraying conditions include: spraying pressure of 0.3 MPa, spraying height of 50 cm, and continuous spraying time of 4 s for each unit. After spraying, the mixture is allowed to stand and absorb at 20 °C for 1 h to obtain a composite powder. S5. Place the composite powder in a freeze-drying tray and spread it out to form a 2cm thick layer. Pre-freeze it for 3 hours at a temperature of -50℃ and a pressure of normal pressure. Then, freeze-dry it for 10 hours at a temperature of -10℃ and a vacuum of 10Pa. Finally, freeze-dry it for 4 hours at a temperature of 25℃ and a vacuum of 5Pa to obtain freeze-dried powder. S6. The freeze-dried powder is pulverized using a pulverizer and sieved using a sieve with a pore size of 0.15 mm. The sieve material is collected and then three-dimensionally rotary mixed using a high-efficiency mixer at a speed of 24 rpm for 8 hours to obtain a standard quality control sample of selenium form in nut matrix. The standard quality control sample of selenium form in nut matrix is ​​sealed in a glass bottle and stored in a refrigerator (0~4℃).

[0040] Test Example 1 This test example determines the content of selenium forms (selenite and selenomethionine) in the sample, as follows: (1) Test sample: Selenium speciation standard control sample in nut matrix prepared in Example 1.

[0041] (2) Reagents and selenium-based standard substances: Reagents: The water used in the experiment met the Class I water requirements in GB / T6682-2008 "Specifications and Test Methods for Water Used in Analytical Laboratories"; citric acid (analytical grade), ammonia (analytical grade), sodium hexanesulfonate (analytical grade), methanol (chromatographic grade), and streptoprotein E; Selenium speciation standard substances: selenite standard solution (GBW10032, National Institute of Metrology, China), selenomethionine (purity >99wt%, Acros Organics).

[0042] (3) Instruments and equipment: ultrasonic cleaner (KQ2200E, Kunshan Ultrasonic Instrument Co., Ltd.), liquid chromatography-inductively coupled plasma tandem mass spectrometer (HPLC-ICP-MS / MS, model 8900, Agilent Technologies).

[0043] (4) Instrument conditions: Chromatographic conditions: Agilent ZORBAX SB-Aq reversed-phase ion-pair column (250 mm × 4.6 mm, 5 μm); mobile phases included mobile phase A and mobile phase B; mobile phase A: an aqueous methanol-water mixture of citric acid and sodium hexanesulfonate, pH unadjusted, with citric acid concentration of 10.00 mmol / L, sodium hexanesulfonate concentration of 5.00 mmol / L, and methanol volume fraction of 2%; mobile phase B: an aqueous methanol-water mixture of citric acid and sodium hexanesulfonate, pH adjusted to 7.00 with ammonia, with citric acid concentration of 10.00 mmol / L, sodium hexanesulfonate concentration of 5.00 mmol / L, and methanol volume fraction of 2%; flow rate: 1.50 mL / min; column temperature: 25 °C; injection volume: 10 μL. ICP-MS / MS conditions: RF power 1500W; plasma gas (Ar) flow rate 15L / min; reaction gas (O2) flow rate 0.8mL / min; Rpq value 0.6; nebulizer gas flow rate 1.1L / min; SeO2 collection. 96+ Isotope data.

[0044] (5) Sample pretreatment method: Accurately weigh about 0.1g of sample into a 15mL centrifuge tube, add 10mL of 50M Tris / HCl buffer and 10mg of streptomycin E, mix well in a vortex mixer, place the centrifuge tube into an ultrasonic extractor, extract for 5h at a temperature of 40~50℃, after extraction, centrifuge at 8000rpm for 3min, filter through a 0.45μm filter membrane, and perform instrument analysis.

[0045] Figure 1 This is the chromatogram of the standard solution. Figure 2 The chromatogram is of the selenium speciation standard control sample in the nut matrix prepared in Example 1. The results show that the chromatographic peaks of selenite and selenomethionine in the selenium speciation standard control sample in the nut matrix prepared in Example 1 have good peak symmetry. Compared with the chromatographic peaks of the standard, it can be seen that the chromatographic peaks of each target analyte and other impurity chromatographic peaks (non-target chromatographic peaks) have good separation, which fully meets the quality requirements of the quality control sample.

[0046] Test Example 2 This test example measures the uniformity of the sample, as follows: Ten samples were randomly selected from the selenium speciation standard control samples of nut matrix packaged in glass bottles prepared in Example 1. The selenite and selenomethionine contents of each sample were determined independently and in parallel twice under repeat conditions.

[0047] Table 2 shows the results of the sample homogeneity test and calculation, specifically using one-way ANOVA (analysis of variance). F Uniformity is tested and evaluated using the test method. F The value is calculated using equations 1 through 3: Formula 1; Formula 2 Formula 3; in, r For each group of measurements, This is the average of the first group within the group. This is the overall average. This is the group average. For a single measurement; K 1 represents the degrees of freedom between groups. K 2 represents the degrees of freedom within the group; Q 1 represents the sum of squares between groups. Q 2 represents the within-group sum of squares, with 9 degrees of freedom and a significance level of 2. α =0.05, the critical value of degrees of freedom can be found in the table. F 0.05 (9,10) = 3.02.

[0048] Calculations and statistics show that: selenite ions F =2.50, selenomethionine F =2.78, statistical calculation of selenite and selenomethionine. F The value is less than the critical value (3.02), and the sample meets the homogeneity requirements, indicating that the content of selenium forms in the sample is uniform.

[0049] Table 2. Sample homogeneity test and calculation results (mg / kg)

[0050] Test Example 3 This test example examines the stability of samples meeting homogeneity requirements. The stability test includes two dimensions: long-term stability (storage stability, conducted at 4℃) and short-term stability (transport stability, conducted at 30℃). Sampling follows a principle of dense sampling followed by sparse sampling. Six sampling time points are set for both short-term and long-term stability testing. Three well-packaged samples are randomly selected from each sampling time point, and each sample undergoes two parallel determinations. The average of the six determinations for the three samples is used to analyze the content of selenite and selenomethionine. In accordance with the "JJF 1343-2022 Standard Reference Material Determination and Homogeneity and Stability Assessment," this test example uses the t-test as the statistical method for stability testing. A curve showing the change of characteristic values ​​over time is plotted to assess whether there is a unidirectional trend in the sample's characteristic values. A linear fitting model is used to quantitatively assess the stability of the standard samples, and a stability trend analysis is performed based on the model calculation results: when the |β1| value of a certain index is less than the critical value t· S ( β 1) When the index remains stable during the observation period, it indicates that the sample has good consistency. The specific calculation formulas are shown in Equations 4 to 6: Equation 4; Formula 5; Formula 6; in: For time intervals, This is the average value over the time interval. These are actual measured values. This is the average of the actual measured values. β 1 represents the slope estimate of the straight line fitted by linear regression; S r The remaining standard deviation; S ( β 1) represents the standard error of the slope. To represent the prediction at time points based on the linear fitting model eigenvalues.

[0051] Table 3 shows the stability results of the selenite simulated sample during transportation at 30℃, and Table 4 shows the stability results of the selenomethionine simulated sample during transportation at 30℃. Therefore, after 7 days of observation at 30℃, the stability of selenite and selenomethionine... β The values ​​of 1 and 2 are 0.0031 (less than the critical value t·).S ( β 1) = 0.0039) and 0.0028 (less than the critical value t· S ( β 1) = 0.0044), confirming that both indicators showed good stability during transportation.

[0052] Table 3. Stability results of simulated selenite samples during transportation at 30℃.

[0053] Table 4. Stability results of selenomethionine simulated samples during transportation at 30℃.

[0054] Table 5 shows the stability results of the selenite simulated sample stored at 4℃ for a long period, and Table 6 shows the stability results of the selenomethionine simulated sample stored at 4℃ for a long period. Therefore, it can be seen that after 12 months of storage at 4℃, the stability of selenite and selenomethionine... β The values ​​of 1 and 2 are 0.00040 (less than the critical value t·). S ( β 1) = 0.0025) and 0.0019 (less than the critical value t· S ( β 1) = 0.0061), confirming that both indicators showed good stability during the storage period.

[0055] Table 5. Stability results of simulated selenite samples stored at 4℃ for a long period of time.

[0056] Table 6. Stability results of selenomethionine simulated samples stored at 4℃ for a long period of time.

[0057] Test Example 4 This test example, based on the technical specifications of JJF 1343-2022 "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials," determined the standard value of selenium speciation in the nut matrix prepared in Example 1. Specifically, a multi-laboratory joint assignment method was employed, involving eight accredited testing institutions (including national research institutes, universities, and certified third-party commercial testing laboratories) to jointly conduct the assignment of the standard control sample. During data processing, the original test data were first subjected to a normality test; then, the Grubbs test was used to identify and remove outliers within each laboratory; and the Cochran test was used to evaluate the equiprecision characteristics of the measurement data between laboratories. Only when all statistical tests met the specified requirements were the relevant data considered statistically representative and included in the final statistical analysis of the assignment results.

[0058] Table 7 shows the interlaboratory test data for standard quality control samples (selenite), and Table 8 shows the interlaboratory test data for standard quality control samples (selenomethionine). The Shapiro-Wilke test was used for normality testing. From the tables, W(n,p) = 0.767 (where n = 3, p = 0.95). The results for selenite and selenomethionine from each laboratory in Tables 7 and 8 are greater than 0.767, therefore the test data from each laboratory can be considered normally distributed. The Grubbs test was used to examine whether there were any questionable values ​​within each laboratory group. From the Grubbs critical value table, λ(0.05,3) = 1.155. The results for selenite and selenomethionine from each laboratory in Tables 7 and 8 show the maximum residual value (…). Umax The absolute values ​​of all values ​​are less than λ(0.05,3)×S, indicating that there are no outliers or suspicious values ​​among the laboratory's values. Therefore, all data are retained and can be used for statistical analysis of the values. The Cochrane test is used to determine whether the laboratory's values ​​are of equal precision. The Cochrane test requires that the C-value (Cochrane test statistic) be less than or equal to C(α,m,n), indicating that the average values ​​of each group of data are of equal precision; otherwise, they are considered outliers and should be removed when calculating the values. Referring to the criticality table, C(0.05,8,3)=0.516. The results of selenite and selenomethionine from each laboratory in Tables 7 and 8 show C-values ​​of 0.2510 and 0.3462, respectively, which are less than the critical value. Therefore, the laboratory test results are of equal precision among the groups, and all data should be retained and can be used for statistical analysis of the values.

[0059] Table 9 shows the average values ​​of selenite and selenomethionine from eight laboratories, which were 0.802 mg / kg and 0.500 mg / kg, respectively. These results were used for result determination.

[0060] Table 7. Interlaboratory test data for standard quality control samples (selenite).

[0061] Table 8. Interlaboratory test data for standard quality control samples (selenomethionine)

[0062] Table 9. Average results of selenite and selenomethionine from 8 laboratories

[0063] After removing suspicious values ​​and performing equal precision tests on the values ​​obtained from each laboratory, further significance and normality tests were conducted on the average values ​​of each laboratory. Statistical analysis showed no significant difference among the average values ​​of the eight laboratories (p>0.05), and the values ​​conformed to a normal distribution (p>0.05), indicating that the data met the requirements for statistical analysis. Therefore, the standard values ​​of the selenium speciation standard control samples in the nut matrix prepared in Example 1 of this invention were determined using the arithmetic mean of the average values ​​from each laboratory, with the standard values ​​for selenite (as Se) and selenomethionine (as Se) set at 0.802 mg / kg and 5.00 mg / kg, respectively.

[0064] According to JJF 1343-2022 "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials", the uncertainty of the assignment result of a standard reference material consists of three parts: the uncertainty caused by the inhomogeneity of the standard reference material. U bb Uncertainty caused by the instability of standard substances U sts Uncertainty caused by the standard reference determination process U char The combined uncertainty can be determined by calculating the uncertainty of each component. U （y） At a confidence level of 95%, the expanded uncertainty is... U The combined uncertainty is twice that of the combined uncertainty. U （y） ,in U （y） as well as U Calculate according to Equations 7 and 8: Formula 7; × Formula 8; Uncertainty caused by inhomogeneity of standard materials U bb The calculation method is shown in Equation 9: Equation 9; in, s bb This represents the standard deviation between bottles. MS between The mean square of the bottles (the variance between groups) is given. MS within The mean square of the bottle (within-group variance). n The number of times each bottle was measured.

[0065] Calculations of selenite and selenomethionine U bb The results were 0.03 mg / kg and 0.079 mg / kg, respectively.

[0066] U sts The calculation can be performed according to the formula shown in Equation 10: Formula 10; Where R is the interlaboratory range (maximum value - minimum value).

[0067] Calculations of selenite and selenomethionine U sts The results were 0.0273 mg / kg and 0.0808 mg / kg, respectively.

[0068] U char The calculation can be performed according to the formula shown in Equation 11: Formula 11; The S-value is the standard deviation of the mean of the values ​​obtained from the eight laboratories. P The value represents the total number of laboratories participating in the value setting, specifically 8.

[0069] Calculations of selenite and selenomethionine U char The results were 0.011 mg / kg and 0.028 mg / kg, respectively.

[0070] Based on the above results, the total synthesis uncertainty of selenite can be calculated as follows: U =0.068 mg / kg (relative uncertainty 4.2%), the total synthesis uncertainty of selenomethionine is U =0.18 mg / kg (relative uncertainty 1.8%), the characteristic value of selenite in the nut matrix prepared in Example 1 of this invention is 0.802±0.068 mg / kg; the characteristic value of selenomethionine is 5.00±0.18 mg / kg.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a standard quality control sample of selenium speciation in a nut matrix, characterized in that, Includes the following steps: The kernel of the abalone fruit is first crushed to obtain kernel particles; The kernel particles are sequentially degreased and then pulverized to obtain degreased powder. A selenite solution was sprayed onto the degreased powder to obtain a composite powder; The composite powder was freeze-dried, then pulverized and mixed in sequence to obtain the standard quality control sample of selenium speciation in the nut matrix.

2. The preparation method according to claim 1, characterized in that, The degreasing agent used in the degreasing treatment includes petroleum ether, and the ratio of the kernel granules to the degreasing agent is 1g:5~8mL; the temperature of the degreasing treatment is 15~30℃; the degreasing treatment is carried out under oscillation conditions, the oscillation rate is 150~180rpm, and the oscillation time is 8~12h; the degreasing treatment and the second pulverization further include: allowing the liquid obtained after the degreasing treatment to stand and then separating the solid and liquid in sequence, and collecting the solid material; the standing time is 25~35min.

3. The preparation method according to claim 1 or 2, characterized in that, The second pulverization process further includes: sieving the material obtained after the second pulverization, and collecting the undersize material as the degreased powder; the sieve used for sieving has an aperture of 0.250~0.425mm.

4. The preparation method according to claim 1, characterized in that, The concentration of the selenite solution is 0.2~2 mg / L, and the ratio of the defatted powder to the selenite solution is 1~3 g: 1 mL; The preparation method of the composite powder includes the following steps: laying the defatted powder to form a layer, spraying the selenite solution onto the layer in the form of a spray, and allowing it to stand for absorption to obtain the composite powder; The thickness of the layer is 0.3~0.6mm; the spraying pressure is 0.2~0.4MPa and the height is 40~60cm; the static absorption temperature is 15~30℃ and the time is 1~3h.

5. The preparation method according to claim 1, characterized in that, The freeze-drying step includes: laying the composite powder to form a layer, and then performing pre-freezing, first freeze-drying and second freeze-drying in sequence; The thickness of the layup is 2-3 cm; the pre-freezing temperature is -40 to -60°C, the pressure is atmospheric pressure, and the time is 2-6 h; the first freeze-drying temperature is -10 to -30°C, the vacuum degree is 5-15 Pa, and the time is 8-16 h; the second freeze-drying temperature is 20-40°C, the vacuum degree is 3-8 Pa, and the time is 3-6 h.

6. The preparation method according to claim 1, characterized in that, The process between the third crushing and the mixing includes: sieving the material obtained after the third crushing and collecting the undersize material; the sieve used for sieving has an aperture of 0.075~0.180mm.

7. The preparation method according to claim 6, characterized in that, The mixing method is three-dimensional rotational mixing, with a rotational speed of 18~30 rpm and a time of 6~10 hours.

8. The standard quality control sample of selenium speciation in nut matrix prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the selenium speciation standard control sample in the nut matrix as described in claim 8 in the detection of selenite content and / or selenomethionine content in plant samples.

10. The application according to claim 9, characterized in that, The plant-based samples include nut samples.

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