Standard substance for detecting cadmium content in rice and preparation method thereof

Abstract

The application discloses a standard substance for detecting cadmium content in rice and a preparation method thereof, and belongs to the technical field of detection. The preparation method directly uses fresh rice as a raw material, introduces a target amount of a shielding element into the rice powder through crushing, adding and freeze-drying, and effectively fixes the shielding element in the rice powder. Due to the use of a specific adding reducing agent, the prepared product can realize customization of the cadmium content, and the product has excellent uniformity and stability, and can be directly applied to instrument calibration, analysis method evaluation, measurement process quality control and capability verification in the process of quantitative detection of cadmium in rice.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a standard substance for detecting cadmium content in rice and its preparation method. Background Technology

[0002] Cadmium is a harmful heavy metal element that is widely present in nature. Because cadmium intake can have a significant impact on the human body, the detection of cadmium levels in the food industry is of paramount importance. Among crops, rice has a strong ability to accumulate cadmium, making it a common grain with a relatively high cadmium content. Surveys show that rice frequently exceeds the permitted levels of cadmium.

[0003] Currently, the detection of cadmium content in rice mainly involves grinding the rice into powder and then using methods such as graphite furnace atomic absorption spectrometry (GB 5009.15-2014), inductively coupled plasma mass spectrometry (ICP-MS), and inductively coupled plasma atomic emission spectrometry (ICP-AES) (GB 5009.268-2016) for detection. The standard samples required for instrument calibration, analytical method evaluation, measurement process quality control, and proficiency testing are primarily cadmium-based reference materials prepared from naturally grown cadmium-positive rice samples. However, these naturally grown standards exhibit significant randomness; the expected concentration of cadmium content is difficult to guarantee, and there are large concentration differences between different batches of samples. This not only makes it difficult to meet customized requirements but also presents problems with uniformity and stability. Summary of the Invention

[0004] Based on the shortcomings of existing technologies, the purpose of this invention is to provide a method for preparing a standard substance for detecting cadmium content in rice. This method uses fresh rice as a direct raw material and introduces the target amount of cadmium through crushing, spiking, and freeze-drying, effectively fixing it in rice powder. The prepared product not only allows for customization of cadmium content but also exhibits excellent uniformity and stability. It can be directly applied to instrument calibration, analytical method evaluation, measurement process quality control, and proficiency testing in the quantitative detection of cadmium in rice.

[0005] A method for preparing a standard substance for detecting cadmium content in rice includes the following steps:

[0006] (1) Fresh rice is crushed, sieved, and dried to obtain rice flour;

[0007] (2) Mix rice flour with standard solution evenly to obtain spiked mixture; the standard solution includes a reducing agent and a designed amount of cadmium-containing substance, wherein the reducing agent is at least one of β-mercaptoethanol, dithiothreitol, and thioglycolic acid, and the concentration is 1-10 mmol / L.

[0008] (3) After freeze-drying the spiked mixture, it is crushed, sieved, and dried until the moisture content is ≤5wt%. Then it is mixed and sterilized to obtain the standard substance for detecting cadmium content in rice.

[0009] In existing technologies, cadmium-containing rice standards are mainly based on native rice varieties. However, to ensure the uniformity and stability of the standards, this invention uses fresh, normal rice as raw material and prepares the product through a post-spikeing process. Research has found that cadmium in rice mainly exists in the form of cadmium-binding proteins containing thiol groups. However, these thiol groups in rice flour are unstable and easily oxidized to form disulfide bonds, especially after rice is pulverized, resulting in a large number of protein thiol groups being oxidized. This prevents cadmium-containing substances from stably binding with rice flour, leading to poor uniformity and stability of the cadmium standard prepared by direct spiked methods. Therefore, this invention effectively reduces the disulfide bonds already formed in the rice flour to thiol groups by diluting a designed amount of cadmium-containing substance and adding a specific type and amount of reducing agent to the diluent. The semi-finished product is then dried using a rapid freeze-drying method to ensure the uniformity and stability of the final product.

[0010] Preferably, the cadmium content of the fresh rice in step (1) is ≤0.05mg / kg.

[0011] Preferably, the mesh size of the sieve during sieving in step (1) is 20 to 200 mesh.

[0012] Preferably, the water content of the rice flour in step (1) is ≤5wt%.

[0013] Before mixing with the standard solution, reducing the water content in rice flour can effectively ensure the full action of the reducing agent and the full loading of cadmium-containing substances.

[0014] Preferably, the cadmium-containing substance is a cadmium solution standard substance.

[0015] Preferably, in step (2), the mass ratio of the standard solution to rice flour is (0.5-1.8):1.

[0016] More preferably, the mass ratio of the standard solution to rice flour is (1-1.5):1.

[0017] Preferably, the reducing agent is β-mercaptoethanol, and its concentration in the standard solution is 4–6 mmol / L.

[0018] Furthermore, the inventors discovered that using β-mercaptoethanol as a reducing agent yields the best results, ensuring that the disulfide bonds in rice flour are effectively reduced to thiol groups, which is beneficial for the binding of cadmium to thiol-containing proteins.

[0019] Preferably, in step (2), the rice flour and the standard solution are mixed by stirring for 5 to 30 minutes, more preferably 8 to 15 minutes.

[0020] Preferably, in step (3), freeze drying includes freeze drying and sublimation drying in sequence. The temperature during the pre-freeze drying is -10 to -70°C and the drying time is 8 to 40 hours. The temperature during the sublimation drying is 10 to 40°C and the drying time is 8 to 40 hours.

[0021] Under the freeze-drying process, rice flour can fully combine with cadmium-containing substances without oxidation. However, if the mixture is dried by other means, the degree of binding will be reduced, or even severe oxidation of the rice flour may occur.

[0022] Preferably, the mesh size of the sieve during sieving in step (3) is 20 to 200 mesh.

[0023] Preferably, the temperature during mixing in step (3) is 18-25°C, the relative humidity is ≤40%, and the mixing time is 10-60h.

[0024] Preferably, sterilization in step (3) is performed by radiation, wherein the radiation dose is 8-12 kGy and the radiation time is 0.5-1.5 h.

[0025] Another object of the present invention is to provide a standard substance for detecting cadmium content in rice prepared by the aforementioned preparation method.

[0026] The standard material for detecting cadmium content in rice described in this invention has good uniformity and stability, and its cadmium content is designed to be controllable. It can be used for instrument calibration, analytical method evaluation, measurement process quality control, and proficiency testing for the determination of cadmium content in similar matrix samples such as grains and their products. In particular, for the verification of detection accuracy, the product described in this invention can be customized for certain concentration points that need to be focused on during the verification of detection accuracy.

[0027] The beneficial effects of this invention are that it provides a standard substance for detecting cadmium content in rice and its preparation method. This method uses fresh rice as a direct raw material and introduces the target amount of cadmium into the rice powder through crushing, spiking, and freeze-drying. Due to the use of a specific spiking reducing agent, the prepared product can not only achieve customization of cadmium content, but also has excellent uniformity and stability. It can be directly applied to instrument calibration, analytical method evaluation, measurement process quality control, and proficiency testing in the quantitative detection of cadmium in rice. Attached Figure Description

[0028] Figure 1This is a standard curve diagram of the standard substance standard solution of the product in Example 1 of the present invention.

[0029] Figure 2 This is a trend chart showing the long-term stability monitoring of the product in Embodiment 1 of the present invention.

[0030] Figure 3 This is a short-term stability monitoring trend chart of the product in Embodiment 1 of the present invention. Detailed Implementation

[0031] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments and comparative examples. The purpose of this description is to provide a detailed understanding of the invention, not to limit its scope. All other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of this invention. Unless otherwise specified, the experimental reagents and instruments involved in the implementation of this invention are commonly used reagents and instruments.

[0032] In a specific embodiment of the present invention, the following instruments are used for the preparation and verification of standard samples:

[0033] Inductively coupled plasma mass spectrometry (ICP-MS / MS) (Agilent Technologies, USA); graphite furnace atomic absorption spectrometry (Agilent Technologies, USA); microwave digestion system (Milstone, Italy); electrically heated drying oven (Shanghai Yiheng Company); freeze dryer (Yonghe Chuangxin Company); high-efficiency mixer (Guangzhou Degong Machinery Equipment Company); electronic balance (Mettler Corporation, USA); Karl Fischer moisture analyzer (Mettler Corporation, USA); ultrapure water system (Milliber-Tech, USA). All the above instruments and equipment are commercially available products. Those skilled in the art can select suitable instruments and equipment according to their actual needs; specific product models are not listed here.

[0034] Example 1

[0035] An embodiment of the standard substance for detecting cadmium content in rice and its preparation method according to the present invention includes the following steps:

[0036] (1) Fresh rice (commercially available rice with cadmium content ≤5mg / kg) is pulverized using a grinder, passed through a 50-mesh sieve, and dried until the moisture content is ≤5wt% to obtain rice flour;

[0037] (2) Mix rice flour and standard solution at a mass ratio of 1:1.2 and stir for 10 minutes until homogeneous to obtain a spiked mixture; the standard solution includes a reducing agent and a designed amount of cadmium solution standard material (the cadmium solution standard material uses certified standard materials approved and promulgated by the national metrology administration department). The standard solution is prepared by first diluting the cadmium solution standard material with water, and then adding the reducing agent β-mercaptoethanol. The concentration of β-mercaptoethanol in the standard solution is 5 mmol / L.

[0038] (3) Prepare a 10mm sample from the soft paste-like spiked mixture, then pre-freeze-dry it at -50℃ for 24h, then sublimate-dry it at 25℃ under a vacuum of 0.001Pa for 24h, crush it, pass it through a 50-mesh sieve, and dry it until the moisture content is ≤5wt%. Then mix it at 20℃ and 40% relative humidity for 48 hours. Sterilize the sample by irradiating it with a cobalt radiation device at a radiation dose of 10kGy for 1h to obtain the standard substance for detecting cadmium content in rice.

[0039] Verify the product:

[0040] (a) Weigh 0.3 g (accurate to 0.001 g) of the prepared solid sample into the microwave digestion vessel, add 5 mL of nitric acid, cover and let stand for 1 hour, then add 5 mL of primary water, tighten the lid, and digest according to the standard operating procedure of the microwave digester. After cooling, remove the vessel, slowly open the lid to release the air, rinse the inner lid with a small amount of water, dilute to a 50 mL volumetric flask with water, mix well and set aside. At the same time, perform a blank test.

[0041] Microwave digestion conditions: programmed temperature rise, heating to 80℃ in 3 minutes, continuously heating to 190℃ within 9 minutes, and maintaining the temperature for 25 minutes for digestion;

[0042] (b) Accurately transfer 0.05 mL of 1000 mg / L standard solution into 500 mL volumetric flasks to prepare 0.1 μg / mL intermediate standard stock solutions. Then, take 0.10, 0.25, 0.50, 1.0, 2.5, and 5.00 mL of the intermediate stock solutions into 50 mL volumetric flasks to prepare working curve solutions of 0.2, 0.5, 1, 2, 5, and 10 μg / L, and analyze them by ICP-MS.

[0043] Inductively coupled plasma mass spectrometry: Acquisition mode: He mode, Detection method: Automatic, Physicochemical chamber temperature: 2℃, Sampling depth: 10mm, RF power: 1550w, Auxiliary gas flow rate: 1.00L / min, Nebulizing gas flow rate: 1.00L / min, Peristaltic pump: 0.1rps, Plasma gas flow rate: 15.0L / min;

[0044] A standard curve was prepared based on the standard solution and the counts per second (CPS) as follows: Figure 1 As shown, the linear range is 0.2–10 μg / L, and the linear equation is: y = 903.59x + 39.17. Assuming a rice sample weight of 0.3g, the linear range for cadmium content in rice flour is 0.03 mg / kg–1.67 mg / kg, with a correlation coefficient of R0. 2 =0.9998;

[0045] (c) Measure the blank sample 10 times consecutively, calculate the mean and standard deviation (SD) of the blank sample, the limit of detection is 3 times the SD, and the limit of quantitation is 10 times the SD.

[0046] The results of the method detection limit and quantitation limit are shown in Table 1. Ten measurements were taken of the blank sample (unspecified rice flour). The calculated average value of the blank sample was 0.026 μg / L and the standard deviation was 0.0035 μg / L. The lowest detection limit was found to be 3 × SD = 3 × 0.0035 μg / L = 0.0105 μg / L, and the quantitation limit was 10 × SD = 10 × 0.0035 μg / L = 0.035 μg / L. The sample volume was adjusted to 50 mL, and the sample amount was calculated as 0.3 g. Therefore, the detection limit of the sample was 0.0105 μg / L × 50 mL / 0.3 g = 0.0018 mg / kg, and the quantitation limit was 0.035 μg / L × 50 mL / 0.3 g = 0.0058 mg / kg.

[0047] Table 1

[0048]

[0049]

[0050] (d) Accurately weigh 0.3 g of blank rice flour (accurate to 0.0001 g), place it in a microwave tube, add an appropriate amount of cadmium standard solution to make its concentration 0.10 mg / kg, mix well, and then analyze according to step (a). Repeat the measurement 7 times. The precision measurement results are shown in Table 2. The calculated relative standard deviation (RSD) is 0.74%, which meets the requirements of the set value.

[0051] Table 2

[0052]

[0053] (e) Accurately weigh 0.3 g of blank rice flour (accurate to 0.0001 g), place it in a microwave tube, add an appropriate amount of cadmium standard solution to make its concentration 0.10 mg / kg and 0.20 mg / kg, mix well, and then analyze according to (a). The spiked recovery rate of the sample was determined, and the accuracy of the measurement results was evaluated by the spiked recovery rate. The results are shown in Table 3. The spiked recovery rate of cadmium in rice flour was 99.5% to 106.2%, which is between 80% and 110%, and meets the requirements for value determination.

[0054] Table 3

[0055]

[0056] (f) Homogeneity test: In accordance with the relevant requirements of JJF1343 "Assignment of Standard Reference Materials and Evaluation of Homogeneity and Stability", 11 samples were randomly selected for homogeneity testing. Each sample unit was measured three times. The following measurement scheme was adopted, and one-way ANOVA was used to test the homogeneity of the samples.

[0057] Number of repetitions 1: 1-3-5-7-9-11-2-4-6-8-10;

[0058] Number of repetitions 2: 11-10-9-8-7-6-5-4-3-2-1;

[0059] Number of repetitions 3: 2-4-6-8-10-1-3-5-7-9-11;

[0060] The results of the product uniformity test are shown in Table 4. One-way ANOVA was used for statistical analysis to obtain Fo. <F ɑ The product has good uniformity.

[0061] Table 4

[0062]

[0063]

[0064] (g) Stability testing:

[0065] Long-term stability: The product was placed at room temperature in the dark, and two units were randomly selected at 0, 1, 2, 4 and 6 months respectively. Each unit was tested in parallel 3 times to conduct long-term stability tests.

[0066] Long-term stability was assessed using a classic stability evaluation scheme, and the statistical data results are shown in Table 5. Figure 2 As shown, based on the standard deviation of β1, a t-test is used to determine the result: |β1|<t 0.95，n-1 The value of ·s(β1) indicates that the slope is not significant and the product has good long-term stability.

[0067] Table 5

[0068]

[0069] Short-term stability: The product was stored in a 50℃ constant temperature chamber (simulating extreme transportation conditions), and stability tests were conducted on days 0, 1, 3, 5, and 7.

[0070] Short-term stability was assessed using a synchronous stability evaluation scheme, and the statistical data results are shown in Table 6 and... Figure 3 As shown, the test method is the same as for long-term stability, and the test result is |β1|<t. 0.95，n-2The slope of ·s(β1) is not significant, indicating that the product has good short-term stability when the transportation is completed within 7 days at an extreme transportation temperature below 50℃.

[0071] Table 6

[0072]

[0073]

[0074] (h) The cadmium content in the product was determined using two different methods based on inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrometry (FFU). The results of the two methods were then tested for equal precision and consistency of average values ​​using F-test and t-test, respectively. The results are shown in Table 7.

[0075] Table 7

[0076] frequency <![CDATA[P ICP-MS ]]> <![CDATA[P GFAAS ]]> 1 0.2204 0.2186 2 0.2208 0.2197 3 0.2202 0.2166 4 0.2221 0.2184 5 0.2173 0.2245 6 0.2284 0.2167 7 0.2224 0.2212 average value 0.2216 0.2194 Standard deviation 0.0034 0.0028

[0077] The F-test results are as follows:

[0078]

[0079] And f 0.95 (6, 6) = 4.28, 1 / f 0.95 (6, 6) < F < f 0.95 (6, 6) The F-test of the two sets of data passed, indicating that the two sets of data have the same precision.

[0080] The t-test results are as follows:

[0081]

[0082] And t 0.95，12 =2.18, t < t 0.95，12 The t-tests for both sets of data passed, and the average values ​​of the two sets of data were consistent.

[0083] Inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrometry (FFAAS) were used as the determination methods. After verification, no significant difference was found between the two sets of determination data; therefore, the two methods have equal precision. The determination results were taken as the average of the measurement results from the two methods based on different principles, as follows:

[0084]

[0085] In summary, the cadmium standard reference value in the product is 0.2205 mg / kg.

[0086] (i) Further, the uncertainty of the cadmium reference material for the product is evaluated:

[0087] According to the technical specifications, one-way ANOVA was used to assess uniformity because s1 2 >s2 2 Uniformity standard deviation s H It can be calculated using a formula:

[0088]

[0089] In the formula: s bb The standard deviation of uniformity; denoted as , where is the within-group variance; and 'n' is the number of measurements within the group.

[0090] Based on the data in Table 4, the uncertainty introduced by uniformity can be calculated as follows:

[0091] u bb =0.00281mg / kg

[0092] Based on the data in Table 5, the uncertainty u introduced by the long-term stability with a validity period of 6 months is... lts The calculation is as follows:

[0093] u lts =s(β1)·t=0.000259*6=0.001554mg / kg

[0094] Based on the data in Table 6, the uncertainty u introduced by short-term stability sts The calculation is as follows:

[0095] u sts =s(β1)·t=0.000174*7=0.001218mg / kg

[0096] The uncertainty introduced by the constant value includes Type A uncertainty u. A and Type B uncertainty u B ,

[0097] The uncertainties introduced by the two different methods of setting the value include the Type A uncertainty introduced by A.

[0098]

[0099] The Type B uncertainty introduced by the setting process mainly consists of the following four parts: standard solution preparation, sample weighing, and standard curve fitting. The Type B standard uncertainty components introduced by the setting process are shown in Table 8.

[0100] Table 8

[0101]

[0102]

[0103] The Type B standard uncertainty obtained from the data in Table 8 is:

[0104]

[0105] Therefore, the standard uncertainty introduced by the constant value is:

[0106]

[0107] The uncertainty in the synthesis of the cadmium standard reference material in rice flour is:

[0108]

[0109] Taking the amount of silver as k=2, the expanded uncertainty is:

[0110] U CRM =k×u CRM =0.0132mg / kg

[0111] In summary, the cadmium standard material uncertainty of the product described in this embodiment is acceptable.

[0112] Example 2

[0113] An embodiment of the standard substance for detecting cadmium content in rice and its preparation method described in this invention differs from Example 1 only in that the reducing agent is dithiothreitol.

[0114] The product of this embodiment was subjected to the same testing and verification as in Example 1, and the experimental results were similar to those of Example 1.

[0115] Example 3

[0116] An embodiment of the standard substance for detecting cadmium content in rice and its preparation method described in this invention differs from Example 1 only in that the reducing agent is thioglycolic acid.

[0117] The product of this embodiment was subjected to the same testing and verification as in Example 1, and the experimental results were similar to those of Example 1.

[0118] Comparative Example 1

[0119] A standard substance for detecting cadmium content in rice and its preparation method are disclosed. The only difference between this standard substance and Example 1 is that the standard solution does not contain a reducing agent.

[0120] The results of the homogeneity test are shown in Table 9. One-way ANOVA was used for statistical analysis, and the results showed F > F. ɑ The sample uniformity failed, indicating that cadmium could not effectively bind with rice flour without the use of a reducing agent, resulting in poor uniformity of cadmium in the rice.

[0121] Table 9

[0122]

[0123]

[0124] Comparative Example 2

[0125] A standard substance for detecting cadmium content in rice and its preparation method are disclosed. The only difference between this standard substance and Example 1 is that the reducing agent in the standard solution is dithioerythritol.

[0126] The results of the uniformity test are shown in Table 10. One-way ANOVA was used for statistical analysis to obtain Fo. <F ɑ The sample exhibits good homogeneity.

[0127] The results of the long-term stability test are shown in Table 11. A t-test was used to determine the results: Test result |β1|>t 0.95,3 The product's long-term stability is not ideal, indicating that erythritol dithioesterol has relatively weak reducing power, making the binding stability of cadmium with rice flour less stable than that of β-mercaptoethanol. Therefore, β-mercaptoethanol is the preferred reducing agent in this method.

[0128] Table 10

[0129]

[0130]

[0131] Table 11

[0132]

[0133] Comparative Example 3

[0134] A standard substance for detecting cadmium content in rice and its preparation method are disclosed. The only difference between this standard substance and Example 1 is that the concentration of the reducing agent in the standard solution is 15 mmol / L.

[0135] The results of the homogeneity test are shown in Table 12. One-way ANOVA was used for statistical analysis, and the result was F > F. ɑ The sample homogeneity failed, indicating that when the concentration of the reducing agent used is too high, it will cause the deactivation of thiol-containing proteins, resulting in cadmium being unable to bind stably with rice flour.

[0136] Table 12

[0137]

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a standard substance for detecting cadmium content in rice, characterized in that, Includes the following steps: (1) Fresh rice is crushed, sieved, and dried to obtain rice flour; the mesh size of the sieve is 20-200 mesh. (2) Mix rice flour and standard solution evenly to obtain spiked mixture; the standard solution includes a reducing agent and a designed amount of cadmium-containing substance, the reducing agent is β-mercaptoethanol, and the concentration in the standard solution is 4~6 mmol / L; the mass ratio of standard solution to rice flour is (0.5~1.8):1, and the cadmium-containing substance is cadmium solution standard substance; (3) After freeze-drying the spiked mixture, it is crushed, sieved, and dried until the moisture content is ≤5wt%, then mixed and sterilized to obtain the standard substance for detecting cadmium content in rice; in step (3), freeze-drying includes pre-freeze-drying and sublimation drying in sequence. The temperature of pre-freeze-drying is -10~-70℃ and the drying time is 8~40h; the temperature of sublimation drying is 10~40℃ and the drying time is 8~40h.

2. The method for preparing the standard substance for detecting cadmium content in rice as described in claim 1, characterized in that, In step (1), the cadmium content of fresh rice is ≤0.05mg / kg and the moisture content of rice flour is ≤5wt%.

3. The method for preparing the standard substance for detecting cadmium content in rice as described in claim 1, characterized in that, In step (2), the rice flour and standard solution are mixed by stirring for 5 to 30 minutes.

4. The method for preparing the standard substance for detecting cadmium content in rice as described in claim 1, characterized in that, In step (3), the mesh size of the sieve is 20 to 200 mesh.

5. The method for preparing the standard substance for detecting cadmium content in rice as described in claim 1, characterized in that, In step (3), the mixing temperature is 18~25℃, the relative humidity is ≤40%, and the mixing time is 10~60h; sterilization is carried out by radiation, the radiation dose is 8~12kGy, and the radiation time is 0.5~1.5h.

6. The standard substance for detecting cadmium content in rice prepared by the preparation method according to any one of claims 1 to 5.

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