Preparation method of standard substance for analyzing phosphorus component in food

Abstract

The invention discloses a preparation method of a standard substance for analyzing phosphorus components in food, which comprises the following steps: (1) cutting off tendons, tendons, bones, skins and fat from fresh livestock meat or poultry meat, and crushing pure meat to obtain meat paste; (2) weighing a certain amount of meat paste, adding a calculated amount of disodium hydrogen phosphate aqueous solution into the meat paste, mixing, and continuously crushing and stirring until the mixture is uniform; (3) carrying out freeze drying on the standard-added and uniformly-mixed meat paste to obtain meat powder blocks; (4) crushing the meat meal blocks, and collecting freeze-dried powder; screening the freeze-dried powder by using a 60-mesh sieve; (5) detecting the moisture content of the sieved freeze-dried powder, wherein the moisture content is less than 5%; and (6) sub-packaging the freeze-dried powder, sealing the freeze-dried powder into independent packaging units, and performing uniformity test to obtain the phosphorus component analysis standard substance. The phosphorus component analysis standard substance has the advantages of uniform phosphorus content, good short-term and long-term stability and accurate content.

Description

Technical Field

[0001] This invention belongs to the field of phosphorus detection in food, and particularly relates to a method for preparing a standard substance for phosphorus analysis in food. Background Technology

[0002] Phosphorus is the sixth most abundant element in the human body after calcium, accounting for approximately 1% of an adult's body weight. It is essential for bone growth, teeth development, kidney function, and nerve conduction. Phosphorus is a constituent element of ribonucleic acid and deoxyribonucleic acid in all cells, indispensable for the organism's genetic metabolism, growth and development, and energy supply. Phosphorus is also an essential element for all cells, necessary for maintaining cell membrane integrity and performing cellular functions. Phospholipids are the main lipid components of cell membranes and are related to membrane permeability; they promote the breakdown of fats and fatty acids, prevent the accumulation of excessive acid or alkali in the blood, and influence the acid-base balance in plasma and cells. Phosphorus promotes the absorption of substances through cells, stimulates hormone secretion, and is beneficial to nerve and mental activities. Phosphorus stimulates neuromuscular function, causing the heart and muscles to contract rhythmically. Phosphorus helps with cell division and proliferation, as well as the synthesis of nucleoproteins, passing on genetic traits from one generation to the next.

[0003] The phosphorus content varies among different types of food. Foods rich in protein, such as meat, fish, eggs, milk, cheese, beans, and nuts, contain more phosphorus; rice provides about 12% of the phosphorus; and except for bones, meat, poultry, and fish contain 15 to 20 times more phosphorus than calcium.

[0004] Currently, most methods for detecting phosphorus in livestock and poultry meat use traditional chemical methods. The national standard GB / T5009.87-2016, "Determination of Phosphorus in Food," specifies spectrophotometric and inductively coupled plasma atomic emission spectrometry methods for determining phosphorus content in food. Among these methods, the molybdenum blue spectrophotometric method and inductively coupled plasma atomic emission spectrometry are suitable for determining phosphorus in various types of food, while the vanadium molybdenum yellow spectrophotometric method is suitable for determining phosphorus in infant food and dairy products.

[0005] Standard reference materials (SRPs) are substances or materials with one or more sufficiently homogeneous characteristic values ​​that have been determined. As "measuring tools" in the analytical measurement industry, they play an indispensable role in calibrating measuring instruments and devices, evaluating analytical methods, measuring the characteristic values ​​of substances or materials, assessing the operational skills of analysts, and controlling product quality during the production process. As "chemical weights" in relative measurements, SRPs play a crucial role in verifying and ensuring the accuracy of test results.

[0006] Behind the research on the nutritional components and safety of livestock and poultry meat lies the support of efficient and accurate testing technologies. The development and application of reference materials provide strong assurance for quality and safety testing and analysis throughout the entire production process of livestock and poultry products. Stable phosphorus content analysis reference materials are essential key chemical reagents used in conjunction with testing methods.

[0007] Since fresh meat is naturally rich in phosphorus, it can be used directly as a blank standard reference candidate without the addition of phosphoric acid or phosphates. This standard reference uses ISO 23776:2021 "Meat and meat products—Determination of total phosphorus content" (ICP-OES method) to detect the total phosphorus content in meat and meat products. The measured total phosphorus consists of two parts: phosphorus from fresh meat and phosphates, a food additive. The development of this standard reference for phosphorus analysis in meat products can simulate the determination of total phosphorus content in real meat products using the ICP-OES method, and the results can be used to evaluate the rationality of adding phosphates to meat products.

[0008] The following terminology analysis is from the book "Standard Reference Material Quality Control and Uncertainty Assessment" edited by Li Hongmei: Homogeneity refers to the state of having the same structure or composition associated with one or more properties of a substance. If the measurement results fall within the specified uncertainty range by measuring samples of a specific size taken from different packaging units (e.g., bottles, bags, etc.) or from the same packaging unit, the standard substance can be considered homogeneous for the specified property quantity.

[0009] Stability refers to the ability of a reference material to maintain its characteristic properties within a specified range over a specific time interval and storage conditions. Stability testing should be conducted after homogeneity testing, taking into account both long-term stability (e.g., shelf life) and short-term stability (e.g., stability under "transportation conditions"). The long-term stability of this reference material should be assessed using a measurement method with precision not lower than a predetermined value and sufficient sensitivity, ensuring consistency of operating and experimental conditions throughout the stability study. Stability samples of the reference material should be randomly selected from samples aliquoted into the smallest possible packaging units. Statistical tests should be performed on the stability measurement data, using trend analysis or analysis of variance.

[0010] The determination of reference material values ​​involves the accurate measurement of candidate materials that have passed homogeneity testing and the systematic analysis and processing of the measurement data. It is one of the most crucial steps in the development of reference materials. The determination of reference material values ​​is not merely a purely scientific and technological research process; the selection of measurement methods and standards requires comprehensive consideration of existing national (international) standards, the intended use of the reference material, and economic factors. Therefore, the determination of reference material values ​​is a systematic undertaking that reflects the level and value of reference material research.

[0011] As measuring instruments, standard reference materials (SRPs) can reproduce, preserve, and transmit measurement values, ensuring the comparability and consistency of values ​​across different times and spaces. Therefore, it is essential to guarantee the traceability of SRP values, meaning that the values ​​of SRPs can be linked to national or international bases and standards through a continuous comparison chain with a specified uncertainty. This requires that the research and development units and collaborating units possess certain qualifications related to the analyte, that personnel have sufficient experience in analytical measurement and uncertainty analysis, and that measuring instruments, pretreatment equipment, and other measuring instruments, in addition to meeting basic metrological requirements, must also be able to meet the requirements for measuring the analyte. The measurement method should be validated, have complete operating procedures, and be proven accurate and reliable both theoretically and practically. It should be traceable through a continuous comparison chain with a given uncertainty to my country's national metrological bases and standards, international measurement standards, or to the metrological bases and standards of countries or economies that have signed MBA mutual recognition agreements under the framework of the International Bureau of Weights and Measures (BIPM). When the continuity of the traceability chain for the quality value of a reference material cannot be ensured, sufficient evidence or proof of relevance should be provided through detailed evaluation of the measurement process or comparative measurements with other recognized national or international certified reference materials. Summary of the Invention

[0012] The purpose of this invention is to provide a method for preparing a standard reference for phosphorus analysis in food, which facilitates the establishment of chemical traceability for the measurement of livestock and poultry products, provides quality assurance for laboratory analysis and testing, and provides technical support for the quality assurance of livestock and poultry products.

[0013] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention discloses a method for preparing a standard reference for phosphorus analysis in food, comprising the following steps: (1) Remove tendons, bones, skin and fat from fresh meat or poultry, and then grind the pure meat to obtain meat paste; (2) Weigh a certain amount of meat paste, add a calculated amount of disodium hydrogen phosphate aqueous solution to it, mix and continue to grind and stir until uniform; (3) Freeze-dry the spiked and mixed meat paste to obtain meat powder blocks; (4) Crush the meat powder blocks and collect the freeze-dried powder; sieve the freeze-dried powder using a 60-mesh sieve; (5) The moisture content of the sieved freeze-dried powder should be tested and found to be less than 5%; (6) The freeze-dried powder is divided into individual packaging units and sealed. The homogeneity test is then performed to obtain the phosphorus component analysis standard substance.

[0014] Each individual package is numbered, and the number of units to be sampled is determined based on the number of individual package units. A random number of units are then selected for homogeneity testing. If the homogeneity test fails, the preparation process needs to be adjusted and optimized, and the standard material needs to be re-developed until the homogeneity test is passed.

[0015] The homogeneity test was conducted according to the requirements of JJF 1343-2022 "Assignment of Standard Reference Materials and Evaluation of Homogeneity and Stability". When the total number of units N>500, the number of units sampled was >=15. Each sample was tested twice for repeatability. Homogeneity was tested by comparing within-group and between-group variances using one-way ANOVA. After the homogeneity test of the standard samples passed, short-term and long-term stability tests were performed. The short-term stability test lasted for one week to examine the stability of the standard samples under transportation conditions. Long-term stability testing involved monitoring samples stored at room temperature (25°C) away from light for 12 months, following a principle of initial close monitoring followed by sparse monitoring, to determine a reliable shelf life and optimal storage conditions.

[0016] The meat is one of pork, beef or mutton, and the poultry is chicken or duck; the phosphorus content analysis standard substance in the food is one of freeze-dried powders made from pork, beef, mutton, chicken or duck.

[0017] The concentration of disodium hydrogen phosphate is set based on the phosphorus content of the standard substance. The concentration of disodium hydrogen phosphate is calculated by working backwards from the amount of phosphorus to be prepared according to the material balance rule; the preferred concentration is 15.585 to 32.086 g / L.

[0018] Preferably, the freezing temperature of the vacuum freeze dryer is -30 to -60°C, the pre-vacuum degree is 0.1 to 0.4 mbar, the first stage of the freeze drying is set with a vacuum degree of 0.5 to 0.9 mbar, the second stage is set with a vacuum degree of 0.5 to 0.9 mbar, and the total freeze drying time is 20 to 30 hours.

[0019] The crushing speed of the crusher in step (4) is 23000-29000 r / min.

[0020] Compared with the prior art, the present invention has the following technical advantages: This study prepared a standard reference material for the analysis of phosphorus content in food. The phosphorus content was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), and the homogeneity was evaluated using one-way ANOVA. The results met the requirements of F < F0. 0.05（r－1,n－r） The stability was assessed using linear regression trend analysis and the t-test, and the results showed that |b1| < t. （0.95,n－2） ×s (b1) Through collaborative determination by 10 laboratories, experimental results have demonstrated that the phosphorus component analysis standard material of this invention has the advantages of uniform phosphorus content, good short-term and long-term stability, and accurate content, and can be used as a reference standard material for phosphorus component analysis in food.

[0021] This invention will promote the healthy development of my country's animal husbandry industry, provide strong guarantees for improving the quality of livestock and poultry products, and provide strong support for enterprises to go global. At the same time, it also provides technical support for various countries to evaluate and verify newly developed international standard methods. This standard reference material mainly plays the following roles in the quality assurance system for livestock and poultry products: verifying, evaluating, and identifying new technologies and methods; establishing chemical traceability of measurements; and controlling experimental processes. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1 A method for preparing a standard reference for phosphorus analysis in food includes the following steps: 1. Component ratio Fresh pork was selected, and the phosphorus content was prepared according to the formula of 170 mg per 100 g of pork. Based on 1000 g of freeze-dried pork powder, 1700 mg of phosphorus needs to be added. According to the chemical mass principle, the amount of disodium hydrogen phosphate to be added is calculated to be 7792.44 mg.

[0024] 2. Preparation process (1) Remove tendons, shanks, bones, skin and fat from fresh pork, cut the remaining pure pork into small pieces, put them into a high-speed chopper for grinding, set the machine speed to 3700r / min, grind 2000-3000g of material each time, and grind for about 20 minutes to get pork paste. (2) Weigh 5000g of minced pork, add 500mL of a 15.585g / L sodium hydrogen phosphate aqueous solution, mix and put it into a high-speed chopper for crushing. Set the machine speed to 3600r / min, crush 2000-3000g of material each time, and crush and stir for about 35min until uniform. (3) Place the spiked and mixed pork paste into a freeze-drying tray and send it into a vacuum freeze dryer for freeze-drying to obtain meat powder blocks; set the parameters of the vacuum freeze dryer: the pre-vacuum degree is 0.2 mbar, the first stage of freeze-drying is set to 0.7 mbar, the second stage is set to 0.7 mbar, the freezing temperature is shown in Table 1, and the total freeze-drying time is about 25 hours. Table 1 Freeze-drying temperature parameters for Example 1 (4) Crush the meat powder blocks using a high-speed grinder at a speed of 28,000 r / min, using 500-1000 g per batch for 1 minute each time, and collect the freeze-dried powder. Send the freeze-dried powder into a sieve and sieve it using a 60-mesh sieve. If there is too much material on the sieve, it needs to be crushed again and sieved. (5) The sieved freeze-dried powder shall be tested for moisture content in accordance with GB 5009.3-2016 National Food Safety Standard - Determination of Moisture in Food. The moisture content shall be less than 5%. (6) After the moisture content test is qualified, the freeze-dried powder is repackaged and sealed into individual packages. Each individual packaged sample is numbered and the uniformity test, stability test and value determination are completed. After the test is passed, the standard substance for phosphorus analysis in food is obtained.

[0025] Test Experiment Example 1 1. Homogeneity testing and evaluation of phosphorus component analysis standard materials: According to JJF 1343-2022 "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials", the total number of standard reference materials developed in this study is 600 bottles. Fifteen bottles were randomly selected for homogeneity testing (sampling ratio 1 / 40), with two data points collected for each sample. Measurements were performed in a random order, and one-way ANOVA was used to evaluate the homogeneity test results. The minimum sample size for phosphorus homogeneity testing was 0.5g. Stability monitoring was conducted after the initial homogeneity test was passed.

[0026] Table 2. Homogeneity test data of phosphorus content analysis standard substances in food One-way ANOVA was used to test the homogeneity within and between bottles. The homogeneity analysis data for the standard substances are shown in Table 2. From the homogeneity test data in Table 2, it can be seen that at a significance level of α=0.05, with r⁻¹=15⁻¹=14 and nr=30⁻¹⁵=15, F⁻¹... 0.05（14,15） Under the condition of F = 2.42, the phosphorus determination results in food conform to the formula: F A <F 0.05 (14,15). Based on this, it can be determined that there are no significant differences within and between groups of the developed standard material, indicating that the standard material is homogeneous and meets the requirements of national standard materials.

[0027] Based on the fact that the within-group variance is less than the between-group variance, the repeatability variance of the detection method is used to assess the uncertainty introduced by sample homogeneity.

[0028] Uncertainty introduced by homogeneity: U bb = = 2.82 mg / 100 g (In the formula: number of repeated measurements n=2, S1) 2 S2 is the sum of squares between groups. 2 (The sum of squares within the group).

[0029] 2. Stability monitoring and evaluation of phosphorus component analysis standard materials The stability of a reference material refers to its property of maintaining its characteristic values ​​within a specified range under certain time intervals and environmental conditions. Short-term and long-term stability tests were conducted on the developed reference materials. Short-term stability tests were performed at four temperature gradients: -20℃, 0℃, 25℃, and 45℃, for one week to examine the stability of the reference material under transportation conditions. Long-term stability tests were conducted by monitoring samples stored at room temperature (25℃) protected from light to determine a reliable shelf life and optimal storage conditions.

[0030] A. Short-term stability study of standard material Short-term stability monitoring data and statistical results are shown in Table 3. |b1|<t (0.95,3) ×s (b1) This indicates that the developed standard material sample has good stability under extreme temperatures of -20 to 45°C within a one-week monitoring period, meeting the relevant requirements in JJF1343-2022.

[0031] Table 3. Results of short-term stability test and statistical evaluation of phosphorus content standard reference materials in food. B. Long-term stability study of standard materials Long-term stability monitoring data and statistical results are shown in Tables 4 and 5. |b1|<t (0.95,4) ×s (b1) All compounds met the requirements. This indicates that the components in the standard reference do not exhibit long-term variation trends, and the developed standard reference sample has good stability within a 12-month monitoring period, meeting the relevant requirements in JJF 1343-2022.

[0032] Table 4. Long-term stability test data of phosphorus content standard reference materials in food. Table 5. Linear fitting and statistical parameters of long-term stability results of phosphorus content analysis standard references in food. Long-term stability assessment primarily examines the effective storage time of the reference material under estimated environmental conditions to predict its shelf life. As shown in Tables 4 and 5, since phosphorus is relatively chemically stable at room temperature, the estimated environmental conditions are room temperature, protection from light, and dryness. During the 12-month monitoring period, the assessment result for the phosphorus content of the reference material is |b1| < t. (0.95,4) ×s (b1) This indicates that the target components of the standard material show no trend of change during the estimated period.

[0033] Uncertainty about long-term stability: U S =sb1×t=0.0808×12=0.970 mg / 100 g (In the formula: sb1 is the standard deviation of the slope, and t is the expected shelf life.) 3. Constant value In accordance with the value determination requirements of JJF 1343-2022 "Value Determination and Homogeneity and Stability Assessment of Standard Reference Materials", a collaborative value determination was conducted using multiple laboratories to determine the values ​​of standard reference materials for phosphorus analysis in food. The 10 participating laboratories possessed the relevant qualifications and were capable of determining phosphorus values. Each laboratory implemented strict quality control during its analytical process, employing two methods (inductively coupled plasma mass spectrometry and inductively coupled plasma atomic emission spectrometry) to measure the standard reference materials. To ensure the standardization of the value determination, the following requirements were set for the methods and reporting data of the determining laboratories: A. Test method: Use standard methods or other accurate and reliable methods; B. Report 6 independent data points for each component. If two testing methods are used, report 6 independent data points for each. C. Each data point should be reported with three significant figures: in mg / 100g.

[0034] When reporting the results of the value determination analysis, D should also indicate the full name of the analytical method used and the name / number of the standard substance used.

[0035] The data processing steps are as follows: The assigned values ​​are first tested for normality using the Gostino method. After confirming that the data are normally distributed, the Grubbs test is used to test for outliers in the parallel data within each group of laboratories. Then, the Cochran test is used to verify that the assigned values ​​of each laboratory are of equal precision. Finally, the Dixon criterion is used to test for outliers in the mean of each laboratory. The assigned values ​​after evaluation and processing are summarized, and the overall mean and uncertainty are calculated.

[0036] (1) Statistical tests for fixed data - normality test The fixed-value data from 10 laboratories are summarized in Table 6. To examine whether the overall distribution of the data is normally distributed, the D'Agostino test was used to test for normality, given that the total sample size is greater than 50. The calculated statistic Y = 0.6532. At a confidence probability of 95%, Y falls within the range of -2.68 to 1.13, thus passing the normality test.

[0037] Table 6. Phosphorus content determination results from each collaborating laboratory (2) Statistical test of fixed value data - Grubbs test Outlier detection in within-group parallel data from each laboratory—Grubbs' test—involves the following steps: 1) Arrange the data: Sort the measurements in ascending order. The suspicious value is either the minimum or the maximum value; 2) Calculate the mean. x and standard deviation S 3) Calculate the deviation: calculate the difference between the average and the minimum value, and the difference between the maximum value and the average value; 4) Identify a suspicious value; 5) Calculate the residual value: u i = x i - x 6) Determine the significance level: α =0.05; 7) Obtain the critical value from the Zagrubbs table: according to α =0.05, n=6, consult the Grubbs table, and find the critical value by intersecting the rows and columns. l (0.05, 6) =1.887; 8) Compare the calculated values ​​| u i | and critical value l (0.05, 6) S ;9) When | u i | >Critical value l(0.05,6) S At that time, x i It should be removed. As shown in Table 7, calculations show that under the conditions of α = 0.05 and n = 6, |u i |<λ (0.05, 6) S indicates that the collaborative value determination results from the 10 laboratories all passed the test and do not need to be discarded.

[0038] Table 7. Grubbs' test results for phosphorus content determination by each collaborative laboratory. (3) Statistical test of fixed value data - Cochran method To verify whether laboratory-set data have equal precision, the Cochrane method is used. First, calculate... m Groups of data n The variance of the data, then calculate the maximum variance and m The ratio of the sum of variances: Based on the chosen significance level α Number of data sets m Number of repeated measurements n Consult the Cochrane test critical value table to obtain the critical value. c ( α , m , n As shown in Table 7, if C < C ( α , m , n The value indicates that the average values ​​of each group of data have the same precision.

[0039] Table 8. Cochrane test data for phosphorus determination results in each collaborative laboratory. (4) Statistical test of fixed value data - Dixon test Outlier tests for the interlaboratory mean were performed using the Dixon test. The measurement data were arranged in ascending order. X （1） ≤ X （2） ≤…≤ X （7） ≤ X （10）， Calculate according to formulas (2) and (3) r 1 and r 10 : r 1=( X （2） - X（1） ) / ( X （9） - X （1） ), r 10 =( X （10） - X （9） ) / ( X （10） - X （8） );when α =0.05, number of groups n When =10, the critical value f (0.05, 10) =0.530. From 8, we know that... r 1 and r 10 The values ​​are all less than f (α,n) If the value is not specified, all data is retained.

[0040] As shown in Tables 6-9, there were no differences in data within groups; there were no differences in the mean values ​​between groups; the measurement accuracy of each laboratory was the same; and the fixed-value data passed the statistical test and could be summarized and statistically analyzed.

[0041] Table 9. Dixon's test results for the average phosphorus content determination results from each collaborative laboratory. Uncertainty of the value of a standard reference = 3.62 mg / 100 g; (In the formula: This represents the average value across all laboratories. (This represents the overall average, where m is the number of laboratories.) (5) Assignment of standard reference values Based on the uncertainties obtained above, calculate the combined standard uncertainty: U = = = 4.69 mg / 100 g Generally, the coverage factor k=2, determined by the confidence probability and degrees of freedom, resulting in expanded uncertainty: U CRM =k×U=2×4.69=9.38 mg / 100 g In summary, the characteristic value of the phosphorus content standard reference in the food sample of this experiment is expressed as (172±9.38) mg / 100 g. (6) Evaluation of the recovery rate of the preparation of standard substances The assigned values ​​of the standard substances were compared with the theoretical phosphorus content in the standard substance matrix to verify the effectiveness of the preparation process and the accuracy of the assigned values. The results are shown in Table 10. The recovery rate of the standard substance preparation was 104%, and its prepared value basically matched the theoretical value, indicating that the preparation route of the standard substance was accurate and feasible.

[0042] Table 10 Recovery rates of standard reference materials Therefore, this invention selects fresh pork as the standard matrix and, through optimized preparation process, establishes a stable and reliable technical route to prepare a standard substance for phosphorus analysis in pork. The homogeneity and stability of this standard substance were tested using inductively coupled plasma mass spectrometry (ICP-MS), and statistical evaluation was performed using one-way ANOVA and t-tests. The results showed good homogeneity and stability. After collaborative value determination and uncertainty calculation by 10 laboratories, its phosphorus characteristic value can be expressed as (172±9.38) mg / 100 g. Furthermore, the calculated phosphorus preparation value is in good agreement with the theoretical value. The prepared standard substance has advantages such as uniform and stable phosphorus content and accurate characteristic values, and can be used as a reference standard for phosphorus analysis in pork.

[0043] Example 2 A method for preparing a standard reference for phosphorus analysis in food includes the following steps: 1. Component ratio Fresh chicken was selected, and the phosphorus content was prepared according to the formula of 350mg per 100g of chicken. Based on 1000g of freeze-dried chicken powder, 3500mg of phosphorus needs to be added. According to the chemical mass principle, the amount of disodium hydrogen phosphate to be added is calculated to be 16043.27mg.

[0044] 2. Preparation process (1) Remove tendons, shanks, bones, skin and fat from fresh chicken, cut the remaining pure chicken into small pieces, put them into a high-speed chopper for grinding, set the machine speed to 3700r / min, grind 2000-3000g of material each time, grind for about 20 minutes to get chicken paste. (2) Weigh 5000g of chicken paste, add 500mL of disodium hydrogen phosphate aqueous solution with a concentration of 32.086 / L, mix and put it into a high-speed chopper for crushing. Set the machine speed to 3600r / min, crush 2000~3000g of material each time, and crush and stir for about 30min until uniform. (3) Place the spiked and mixed chicken paste into a freeze-drying tray and send it into a vacuum freeze dryer for freeze-drying to obtain meat powder blocks; set the parameters of the vacuum freeze dryer: the pre-vacuum degree is 0.3 mbar, the vacuum degree of the first stage of freeze-drying is set to 0.8 mbar, the vacuum degree of the second stage is set to 0.8 mbar, the freezing temperature is shown in Table 11, and the total freeze-drying time is about 25 hours; Table 11 Freeze-drying temperature parameters for Example 2 (4) Crush the meat powder blocks using a high-speed grinder at a speed of 27,000 r / min, using 500-1000 g per batch for 1 minute each time, and collect the freeze-dried powder. Send the freeze-dried powder into a sieve and sieve it using a 60-mesh sieve. If there is too much material on the sieve, it needs to be crushed again and sieved. (5) The sieved freeze-dried powder shall be tested for moisture content in accordance with GB 5009.3-2016 National Food Safety Standard - Determination of Moisture in Food. The moisture content shall be less than 5%. (6) After the moisture content test is qualified, the freeze-dried powder is repackaged and sealed into individual packages. Each individual packaged sample is numbered and the uniformity test, stability test and value determination are completed. After the test is passed, the standard substance for phosphorus analysis in food is obtained.

[0045] Test Experiment Example 2 1. Homogeneity testing and evaluation of phosphorus component analysis standard materials According to JJF 1343-2022 "Assignment and Homogeneity and Stability Assessment of Standard Reference Materials", the total number of standard reference materials developed in this study is 600 bottles. Fifteen bottles were randomly selected for homogeneity testing (sampling ratio 1 / 40), with two data points collected for each sample. Measurements were performed in a random order, and one-way ANOVA was used to evaluate the homogeneity test results. The minimum sample size for phosphorus homogeneity testing was 0.5g. Stability monitoring was conducted after the initial homogeneity test was passed.

[0046] Table 12. Data on the homogeneity of phosphorus content analysis standard substances in food (Results in mg / 100g) One-way ANOVA was used to test the homogeneity within and between bottles. The homogeneity analysis data for the standard substances are shown in Table 11. From the homogeneity test data in Table 11, it can be seen that at a significance level of α=0.05, with r⁻¹=15-1=14 and nr=30-15=15, F… 0.05（14,15） Under the condition of F = 2.42, the phosphorus determination results in food conform to the formula: F A <F 0.05(14,15). Based on this, it can be determined that there are no significant differences within and between groups of the developed standard material, indicating that the standard material is homogeneous and meets the requirements of national standard materials.

[0047] Based on the fact that the within-group variance is less than the between-group variance, the repeatability variance of the detection method is used to assess the uncertainty introduced by sample homogeneity.

[0048] Uncertainty introduced by homogeneity: U bb = = 13.5 mg / 100 g; In the formula: the number of repeated measurements n=2, S1 2 S2 is the sum of squares between groups. 2 The sum of squares within the group.

[0049] 2. Stability monitoring and evaluation of phosphorus component analysis standard materials The stability of a reference material refers to its property of maintaining its characteristic values ​​within a specified range under certain time intervals and environmental conditions. Short-term and long-term stability tests were conducted on the developed reference materials. Short-term stability tests were performed at four temperature gradients: -20℃, 0℃, 25℃, and 45℃, for one week to examine the stability of the reference material under transportation conditions. Long-term stability tests were conducted by monitoring samples stored at room temperature (25℃) protected from light to determine a reliable shelf life and optimal storage conditions.

[0050] A. Short-term stability study of standard reference material Short-term stability monitoring data and statistical results are shown in Table 13. |b1|<t (0.95,3) ×s (b1) This indicates that the developed standard material sample has good stability under extreme temperatures of -20 to 45°C within a one-week monitoring period, meeting the relevant requirements in JJF 1343-2022.

[0051] Table 13 Results of short-term stability test and statistical evaluation of phosphorus content analysis standard reference materials in food. B. Long-term stability study of standard materials Long-term stability monitoring data and statistical results are shown in Tables 14 and 15. |b1|<t (0.95,4) ×s (b1) All compounds met the requirements. This indicates that the components in the standard reference do not exhibit long-term variation trends, and the developed standard reference sample has good stability within a 12-month monitoring period, meeting the relevant requirements in JJF 1343-2022.

[0052] Table 14 Long-term stability test data of phosphorus content standard reference materials in food Table 15 Linear fitting and statistical parameters of long-term stability results of phosphorus content analysis standard references in food. Long-term stability assessment primarily examines the effective storage time of the reference material under estimated environmental conditions to predict its shelf life. As shown in Tables 14 and 15, since phosphorus is relatively chemically stable at room temperature, the estimated environmental conditions are room temperature, protection from light, and dryness. During the 12-month monitoring period, the assessment result for the phosphorus content of the reference material is |b1| < t. (0.95,4) ×s (b1) This indicates that the target components of the standard material show no trend of change during the estimated period.

[0053] Uncertainty about long-term stability: U S =sb1×t=1.0726×12=12.9 mg / 100 g (In the formula: sb1 is the standard deviation of the slope, and t is the expected shelf life.) 3. Constant value In accordance with the value determination requirements of JJF 1343-2022 "Value Determination and Homogeneity and Stability Assessment of Standard Reference Materials", a collaborative value determination process involving multiple laboratories was adopted to determine the values ​​of standard reference materials for phosphorus analysis in food. The 10 participating laboratories possessed the relevant qualifications and the necessary conditions for phosphorus value determination. Each laboratory implemented strict quality control throughout its analytical process, employing two methods (inductively coupled plasma mass spectrometry and inductively coupled plasma atomic emission spectrometry) to measure the standard reference materials. To ensure the standardization of the value determination, the following requirements were set for the methods and reporting data of the determining laboratories: A. Test method: Use standard methods or other accurate and reliable methods; B. Report 6 independent data points for each component. If two testing methods are used, report 6 independent data points for each. C. Each data point should be reported with three significant figures: in mg / 100g.

[0054] When reporting the results of the value determination analysis, D should also indicate the full name of the analytical method used and the name / number of the standard substance used.

[0055] The data processing steps are as follows: The assigned values ​​are first tested for normality using the Gostino method. After confirming that the data are normally distributed, the Grubbs test is used to test for outliers in the parallel data within each group of laboratories. Then, the Cochran test is used to verify that the assigned values ​​of each laboratory are of equal precision. Finally, the Dixon criterion is used to test for outliers in the mean of each laboratory. The assigned values ​​after evaluation and processing are summarized, and the overall mean and uncertainty are calculated.

[0056] (1) Statistical tests for fixed data - normality test The fixed-value data from 10 laboratories are summarized in Table 16. To examine whether the overall distribution of the data is normally distributed, the D'Agostino test was used to test for normality, given that the total sample size is greater than 50. The calculated statistic Y = -0.9181. At a confidence probability of 95%, Y falls within the range of -2.68 to 1.13, thus passing the normality test.

[0057] Table 16. Phosphorus content determination results from various collaborative laboratories (2) Statistical test of fixed value data - Grubbs test Outlier detection in within-group parallel data from each laboratory—Grubbs' test—involves the following steps: 1) Arrange the data: Sort the measurements in ascending order. The suspicious value is either the minimum or the maximum value; 2) Calculate the mean. x and standard deviation S 3) Calculate the deviation: calculate the difference between the average and the minimum value, and the difference between the maximum value and the average value; 4) Identify a suspicious value; 5) Calculate the residual value: u i = x i - x 6) Determine the significance level: α =0.05; 7) Obtain the critical value from the Zagrubbs table: according to α =0.05, n=6, consult the Grubbs table, and find the critical value by intersecting the rows and columns. l (0.05, 6) =1.887; 8) Compare the calculated values ​​| u i | and critical value l (0.05, 6) S ;9) When | u i | >Critical value l(0.05,6) S At that time, x i It should be removed. As shown in Table 17, calculations show that under the conditions of α = 0.05 and n = 6, |u i |<λ (0.05, 6) S indicates that the collaborative value determination results from the 10 laboratories all passed the test and do not need to be discarded.

[0058] Table 17. Grubbs' test results for phosphorus determination by each collaborative laboratory. (3) Statistical test of fixed value data - Cochran method To verify whether laboratory-set data have equal precision, the Cochrane method is used. First, calculate... m Groups of data n The variance of the data, then calculate the maximum variance and m The ratio of the sum of variances: Based on the chosen significance level α Number of data sets m Number of repeated measurements n Consult the Cochrane test critical value table to obtain the critical value. c ( α , m , n As shown in Table 7, if C < C ( α , m , n The value indicates that the average values ​​of each group of data have the same precision.

[0059] Table 18. Cochrane test data for phosphorus determination results in each collaborative laboratory. (4) Statistical test of fixed value data - Dixon test Outlier tests for the interlaboratory mean were performed using the Dixon test. The measurement data were arranged in ascending order. X （1） ≤ X （2） ≤…≤ X （7） ≤ X （10）， Calculate according to formulas (2) and (3) r 1 and r 10 : r 1=( X （2） - X（1） ) / ( X （9） - X （1） ), r 10 =( X （10） - X （9） ) / ( X （10） - X （8） );when α =0.05, number of groups n When =10, the critical value f (0.05, 10) =0.530. From 8, we know that... r 1 and r 10 The values ​​are all less than f (α,n) If the value is not specified, all data is retained.

[0060] As shown in Tables 16-19, there were no differences in data within groups; there were no differences in the mean values ​​between groups; the measurement accuracy of each laboratory was the same, and the fixed value data passed the statistical test and could be summarized and statistically analyzed.

[0061] Table 19. Dixon's test results for the average phosphorus content determination results from each collaborative laboratory. Uncertainty of the value of a standard reference = 5.00mg / 100g; In the formula: This represents the average value across all laboratories. is the overall average, and m is the number of laboratories.

[0062] (5) Assignment of standard reference values Based on the uncertainties obtained above, calculate the combined standard uncertainty: U = = = 19.3 mg / 100 g Generally, the coverage factor k=2, determined by the confidence probability and degrees of freedom, resulting in expanded uncertainty: U CRM =k×U=2×19.3=38.6 mg / 100 g In summary, the characteristic value of the phosphorus content standard reference in the food sample of this experiment is expressed as (364±38.6) mg / 100 g. (6) Evaluation of the recovery rate of the preparation of standard substances The assigned values ​​of the standard substances were compared with the theoretical phosphorus content in the standard substance matrix to verify the effectiveness of the preparation process and the accuracy of the assigned values. The results are shown in Table 20. The recovery rate of the standard substance preparation was 104%, and its prepared value basically matched the theoretical value, indicating that the preparation route of the standard substance was accurate and feasible.

[0063] Table 20 Recovery rates of standard reference materials Therefore, this invention selects fresh chicken meat as the standard matrix and, through optimized preparation process, establishes a stable and reliable technical route to prepare a standard substance for phosphorus analysis in chicken meat. The homogeneity and stability of this standard substance are detected using inductively coupled plasma mass spectrometry (ICP-MS), and are further analyzed using one-way ANOVA and... t The test method was used for statistical evaluation. The results showed that its homogeneity and stability were good; after collaborative value determination and uncertainty calculation by 10 laboratories, its phosphorus elemental characteristic value can be expressed as (364±38.6) mg / 100g; at the same time, the calculated phosphorus preparation value basically matches the theoretical set value. The prepared standard substance has the advantages of uniform and stable phosphorus content and accurate characteristic value, and can be used as a reference standard substance for the analysis of phosphorus components in chicken meat.

Claims

1. A method for preparing a standard reference for phosphorus analysis in food, characterized in that, Includes the following steps: (1) Remove tendons, bones, skin and fat from fresh meat or poultry, and then grind the pure meat to obtain meat paste; (2) Weigh a certain amount of meat paste, add a calculated amount of disodium hydrogen phosphate aqueous solution to it, mix and continue to grind and stir until uniform; (3) Freeze-dry the spiked and mixed meat paste to obtain meat powder blocks; (4) Crush the meat powder blocks and collect the freeze-dried powder; sieve the freeze-dried powder using a 60-mesh sieve; (5) The moisture content of the sieved freeze-dried powder should be tested and found to be less than 5%; (6) The freeze-dried powder is divided into individual packaging units and sealed. The homogeneity test is then performed to obtain the phosphorus component analysis standard substance.

2. The method for preparing the standard reference for phosphorus analysis in food according to claim 1, characterized in that, The homogeneity test shall be conducted in accordance with the requirements of JJF 1343-2022 "Assessment of Standard Reference Materials and Evaluation of Homogeneity and Stability", when the total number of units N>500, the number of units sampled shall be >=15.

3. The method for preparing the standard reference for phosphorus analysis in food according to claim 1, characterized in that, The meat is one of pork, beef or mutton, and the poultry is chicken or duck.

4. The method for preparing the standard reference for phosphorus analysis in food according to claim 1, characterized in that, The phosphorus content analysis standard material in the food is one of the freeze-dried powders made from pork, beef, mutton, chicken, or duck.

5. The method for preparing the standard reference for phosphorus analysis in food according to claim 1, characterized in that, The concentration of disodium hydrogen phosphate is set based on the phosphorus content of the standard substance. The concentration of disodium hydrogen phosphate is calculated by working backwards from the amount of phosphorus to be prepared, according to the material balance rules.

6. The method for preparing the standard reference for phosphorus analysis in food according to claim 1, characterized in that, The concentration of disodium hydrogen phosphate is 33.92–64.17 g / L.

7. The method for preparing the standard reference for phosphorus analysis in food according to claim 1, characterized in that, The freezing temperature of the vacuum freeze dryer is -30 to -60°C, the pre-vacuum degree is 0.1 to 0.4 mbar, the first stage of the freeze drying is set with a vacuum degree of 0.5 to 0.9 mbar, the second stage is set with a vacuum degree of 0.5 to 0.9 mbar, and the total freeze drying time is 20 to 30 hours.

8. The method for preparing the standard reference for phosphorus analysis in food according to claim 1, characterized in that, The crushing speed of the crusher in step (4) is 23000-29000 r / min.

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