Method for simultaneously determining nine selenium forms in plant selenium-rich food

Abstract

The invention provides a method for simultaneously determining nine selenium forms in plant selenium-rich food, and belongs to the technical field of component analysis. By optimizing enzymolysis conditions and parameters of chromatography-mass spectrometry, high-efficiency extraction, precise separation and high-sensitivity detection of selenium forms are realized, and the method can be used for simultaneously determining nine selenium forms. Wherein the combination, the dosage and the treatment time of the specific proteases can realize efficient extraction of selenium, and the specific proteases show a relatively good synergistic effect, so that an enzymolysis system is optimized; the specific pH value of the mobile phase can change the charged state of the selenium form, induce the molecular configuration change and influence the compound stability, so that the baseline separation of various selenium forms is realized; by setting the specific methanol concentration in the eluent, the good elution efficiency and signal enhancement of each selenium form are ensured, and the gradient elution mode is matched, so that different selenium forms can be efficiently separated at the same time, and various selenium forms can be efficiently and stably detected at the same time.

Description

Technical Field

[0001] This invention relates to the field of component analysis technology, specifically to a method for simultaneously determining nine selenium forms in plant-based selenium-enriched foods. Background Technology

[0002] Selenium is an essential trace element for the human body, possessing important physiological functions such as antioxidation, immune regulation, and maintenance of thyroid function. However, its biological activity and toxicity are closely related to its chemical form. Different selenium forms (such as inorganic selenium, organic selenium, and volatile selenium) exhibit significant differences in absorption, metabolism, and toxicity within organisms. Therefore, establishing accurate detection methods for selenium forms is crucial for assessing the nutritional value and safety of selenium-enriched agricultural products.

[0003] Currently, selenium speciation analysis mainly includes coupled techniques such as liquid chromatography-atomic fluorescence spectrometry (LC-AFS), liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS), gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS), and capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS). However, existing methods typically only detect some selenium speciations (such as selenomethionine and selenocysteine), making it difficult to simultaneously determine multiple speciations such as selenocysteine, selenoethionine, selenolanthiocyanate, and selenourea. Furthermore, the complex matrix of plant-based selenium-enriched foods makes selenium speciation susceptible to influences from extraction conditions and sample pretreatment, limiting the accuracy and repeatability of detection results.

[0004] Therefore, developing an efficient and sensitive detection method that can simultaneously determine multiple selenium forms is of great significance for the quality evaluation, nutritional function research, and safety risk assessment of plant-based selenium-enriched foods. Summary of the Invention

[0005] In view of the technical problems existing in the background art, this application provides a method for simultaneously determining nine selenium forms in plant-based selenium-enriched foods, aiming to solve the problems of low extraction efficiency, difficulty in separating multiple forms, poor repeatability and insufficient sensitivity of existing selenium forms in plant-based selenium-enriched foods.

[0006] This application provides a method for simultaneously determining nine selenium forms in plant-based selenium-enriched foods, comprising the following steps: S1. Crush the plant-based selenium-enriched food sample to obtain selenium-enriched sample powder; S2. Dissolve the selenium-enriched sample powder in a buffer, add protease, sonicate to hydrolyze, and then centrifuge to obtain the test solution; S3. The nine selenium species in the test solution were determined using high performance liquid chromatography-inductively coupled plasma mass spectrometry.

[0007] In the technical solution of this application embodiment, by optimizing the enzymatic hydrolysis conditions and chromatography-mass spectrometry parameters, efficient extraction, accurate separation and high-sensitivity detection of selenium forms are achieved, enabling the simultaneous determination of nine selenium forms: selenocysteine, methylselenocysteine, selenomethionine, selenoethionine, methylselenoic acid, selenolanthiocyanate dihydrochloride, selenate, selenite and selenourea.

[0008] In some embodiments, in step S2, the protease is a breakdown enzyme and a streptin; the mass of the protease accounts for 3-5% of the selenium-enriched sample powder.

[0009] In this embodiment, by combining and using specific proteases, two specific proteases exhibit a good synergistic effect, which can achieve efficient extraction of selenium.

[0010] In some embodiments, in step S3, the conditions for high-performance liquid chromatography (HPLC) in the HPLC-ICP-MS system are as follows: Mobile phase A: 15 mmol / L citric acid + 5 mmol / L sodium hexanesulfonate solution + methanol, wherein the pH of mobile phase A is 3.0, and the volume fraction of methanol in mobile phase A is 1%; Mobile phase B: 15 mmol / L citric acid + 5 mmol / L sodium hexanesulfonate solution; the pH of mobile phase A is 7.2; and the volume fraction of methanol in mobile phase B is 1%. Chromatographic column: Aglient ZORBAX SB-Aq reversed-phase chromatography column (250 mm × 4.6 mm, 5 μm); Column temperature: 20℃; Elution method: gradient elution; The parameters for the gradient elution are shown in the table below:

[0011] In this embodiment, by adjusting the pH value of a specific mobile phase, the charged state of selenium forms is changed, molecular configuration changes are induced, and the stability of the compound is affected. Ultimately, the interaction strength of these selenium forms in the chromatographic system is controlled, ensuring the retention behavior and high resolution of different selenium forms on the chromatographic column, and achieving baseline separation of multiple selenium forms. By setting a specific methanol concentration in the eluent, good elution efficiency and signal enhancement for each selenium form are ensured, while avoiding peak overlap and signal instability caused by a high methanol ratio. Combined with gradient elution, it is possible to simultaneously and efficiently separate different selenium forms and simultaneously perform efficient and stable detection of multiple selenium forms.

[0012] In some embodiments, in step S2, the conditions for inductively coupled plasma mass spectrometry in the high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) system are as follows: Radio frequency power: 1550W; sampling depth: 8.0mm; nebulizer flow rate: 1.03L / min; peristaltic pump speed: 0.3rps; acquisition mode: O2; nebulizer temperature: 2℃; integration time: 0.5s; acquisition time: 540s; reaction gas: oxygen; reaction gas flow rate: 25%; mass numbers of Q1 and Q2: 78 and 94, respectively.

[0013] In this embodiment, oxygen is used as the collision reaction gas. By adjusting the volume fraction of oxygen in the collision reaction cell, the detected electrical signal intensity is optimized and the background equivalent concentration (BEC) value is minimized, which means the instrument has the highest reaction sensitivity.

[0014] In some embodiments, in step S2, the enzymatic hydrolysis time is 38-42 min, the temperature is 37°C, the ultrasonic power is 600W, and the ultrasonic frequency is 40KHz.

[0015] In this embodiment, the extraction efficiency of selenium forms in plant-based selenium-enriched foods is optimal through a specific enzymatic hydrolysis time, and the protease and selenium-enriched sample powder are thoroughly mixed in a buffer solution through ultrasonic treatment.

[0016] In some embodiments, step S2, the centrifugal separation includes the following steps: Centrifuge at 10000 r / min for 15 min, then pass the supernatant through a 0.22 μm aqueous filter membrane to obtain the test solution.

[0017] In this embodiment, the selenium-rich test solution is obtained by centrifugation and filtration.

[0018] In some embodiments, in step S3, the nine selenium forms include selenocysteine, methylselenocysteine, selenomethionine, selenoethionine, methylselenoic acid, selenolanthioamine dihydrochloride, selenate, selenite, and selenourea.

[0019] In this embodiment, the bioavailability and physiological activity of different selenium forms vary greatly. By simultaneously measuring multiple different selenium forms, the actual nutritional value and potential health benefits of selenium-enriched organisms can be more accurately assessed. The health benefits cannot be reliably predicted based solely on the total selenium content.

[0020] In some embodiments, in step S2, the buffer is Tris-HCl, the concentration of the buffer is 75 mmol, and the pH value of the buffer is 7.5.

[0021] In this embodiment, the buffer stabilizes the mobile phase pH, ensuring consistent interaction between the solute and stationary phase during separation. Furthermore, it improves peak shape, enhances resolution and selectivity, and protects the column from pH damage.

[0022] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0023] Figure 1 This is a standard chromatogram of selenium speciation in the test solution in the embodiments of this application.

[0024] Figure 2 This is a chromatogram of nine selenium speciations in the matrix-spiked test solution in the embodiments of this application.

[0025] Figure 3 This is a diagram showing the extraction results of selenium by different proteases in the embodiments of this application.

[0026] Figure 4 This is a graph showing the effect of different protease concentrations on the extraction of selenium speciation in the embodiments of this application.

[0027] Figure 5 The figure shows the extraction results of selenium speciation at different enzymatic hydrolysis times in the embodiments of this application.

[0028] Figure 6 This is a diagram showing the separation results of selenium speciation by different mobile phase pH values ​​in the embodiments of this application.

[0029] Figure 7 The diagram shows the separation results of selenium speciation by methanol at different proportions in the embodiments of this application.

[0030] Figure 8 The images show the detection signals and background intensity results under different O2 contents in the embodiments of this application. Detailed Implementation

[0031] The embodiments of the technical solution of this application are described in detail below. The following embodiments are only used to illustrate the technical solution of this application more clearly, and are therefore only examples, and should not be used to limit the scope of protection of this application.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terms “comprising” and “having” and any variations thereof as used herein are for the purpose of describing particular embodiments only and are not intended to limit this application.

[0033] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0034] To address the problems of low extraction efficiency, difficulty in separating multiple selenium forms, poor repeatability, and insufficient sensitivity of existing selenium-enriched plant-based foods, this application provides a method for simultaneously determining nine selenium forms in these foods. By optimizing enzymatic hydrolysis conditions and chromatography-mass spectrometry parameters, efficient extraction, accurate separation, and high-sensitivity detection of selenium forms are achieved. This method can simultaneously determine nine selenium forms: selenocysteine, methylselenocysteine, selenomethionine, selenoethionine, selenomethionine, selenolanethionine, selenate, selenite, and selenourea. The combination, dosage, and processing time of specific proteases can achieve efficient selenium extraction. These proteases exhibit good synergistic effects, optimizing the enzymatic hydrolysis system, avoiding competitive inhibition between enzymes, and improving extraction efficiency and cost-effectiveness. Specific mobile phase pH values ​​can alter the charged state of selenium speciation, induce molecular conformational changes, and affect compound stability, ultimately regulating their interaction strength in the chromatographic system. This ensures the retention behavior and high resolution of different selenium speciations on the chromatographic column, achieving baseline separation of multiple selenium speciations. By setting a specific methanol concentration in the eluent, good elution efficiency and signal enhancement for each selenium speciation are ensured, while avoiding peak overlap and signal instability caused by high methanol ratios. Combined with gradient elution, this enables simultaneous and efficient separation of different selenium speciations and efficient and stable detection of multiple selenium speciations.

[0035] This application provides a method for simultaneously determining nine selenium forms in plant-based selenium-enriched foods, comprising the following steps: S1. Crush the plant-based selenium-enriched food sample to obtain selenium-enriched sample powder; S2. Dissolve the selenium-enriched sample powder in a buffer, add protease, sonicate to hydrolyze, and then centrifuge to obtain the test solution; S3. The nine selenium species in the test solution were determined using high performance liquid chromatography-inductively coupled plasma mass spectrometry.

[0036] In the technical solution of this application embodiment, by optimizing the enzymatic hydrolysis conditions and chromatography-mass spectrometry parameters, efficient extraction, accurate separation and high-sensitivity detection of selenium forms are achieved, enabling the simultaneous determination of nine selenium forms: selenocysteine, methylselenocysteine, selenomethionine, selenoethionine, selenomethionine, selenolanthionine, selenate, selenite, and selenourea.

[0037] Furthermore, in some embodiments, in step S2, the protease is a breakdown enzyme and a streptin; the mass of the protease accounts for 3-5% of the selenium-enriched sample powder.

[0038] In the technical solution of this application embodiment, by combining and using specific proteases, two specific proteases exhibit a good synergistic effect, which can achieve efficient extraction of selenium.

[0039] Furthermore, in some embodiments, in step S3, the conditions for high-performance liquid chromatography (HPLC) in the HPLC-ICP-MS / MS system are as follows: Mobile phase A: 15 mmol / L citric acid + 5 mmol / L sodium hexanesulfonate solution + methanol, wherein the pH of mobile phase A is 3.0, and the volume fraction of methanol in mobile phase A is 1%; Mobile phase B: 15 mmol / L citric acid + 5 mmol / L sodium hexanesulfonate solution; the pH of mobile phase A is 7.2; and the volume fraction of methanol in mobile phase B is 1%. Chromatographic column: Aglient ZORBAX SB-Aq reversed-phase chromatography column (250 mm × 4.6 mm, 5 μm); Column temperature: 20℃; Elution method: gradient elution; The parameters for the gradient elution are shown in the table below:

[0040] In the technical solution of this application embodiment, by adjusting the pH value of a specific mobile phase, the charged state of selenium forms is changed, molecular configuration changes are induced, and the stability of compounds is affected, ultimately controlling the interaction strength of selenium in the chromatographic system. This ensures the retention behavior and high resolution of different selenium forms on the chromatographic column, achieving baseline separation of multiple selenium forms. By setting a specific methanol concentration in the eluent, good elution efficiency and signal enhancement of each selenium form are ensured, while avoiding peak overlap and signal instability caused by a high methanol ratio. Combined with gradient elution, it is possible to simultaneously and efficiently separate different selenium forms and simultaneously perform efficient and stable detection of multiple selenium forms.

[0041] Furthermore, in some embodiments, in step S2, the conditions for inductively coupled plasma mass spectrometry in the high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) system are as follows: Radio frequency power: 1550W; sampling depth: 8.0mm; nebulizer flow rate: 1.03L / min; peristaltic pump speed: 0.3rps; acquisition mode: O2; nebulizer temperature: 2℃; integration time: 0.5s; acquisition time: 540s; reaction gas: oxygen; reaction gas flow rate: 25%; mass numbers of Q1 and Q2: 78 and 94, respectively.

[0042] In the technical solution of this application embodiment, oxygen is used as the collision reaction gas. By adjusting the volume fraction of oxygen in the collision reaction cell, the detected electrical signal intensity is optimized and the background equivalent concentration (BEC) value is minimized, that is, the instrument's reaction sensitivity is maximized.

[0043] Furthermore, in some embodiments, in step S2, the enzymatic hydrolysis time is 38-42 minutes, the temperature is 37°C, the ultrasonic power is 600W, and the ultrasonic frequency is 40kHz.

[0044] In the technical solution of this application embodiment, the extraction efficiency of selenium form in plant-based selenium-enriched foods is optimal through a specific enzymatic hydrolysis time, and the protease and selenium-enriched sample powder are fully mixed in a buffer by ultrasonic treatment.

[0045] Furthermore, in some embodiments, step S2 includes the following steps of centrifugation: Centrifuge at 10000 r / min for 15 min, then pass the supernatant through a 0.22 μm aqueous filter membrane to obtain the test solution.

[0046] In the technical solution of this application embodiment, a selenium-rich test solution is obtained by centrifugation and filtration separation.

[0047] Furthermore, in some embodiments, in step S3, the nine selenium forms include selenocysteine, methylselenocysteine, selenomethionine, selenoethionine, methylselenoic acid, selenolanthiocyanate dihydrochloride, selenate, selenite, and selenourea.

[0048] In the technical solutions of this application, the bioavailability and physiological activity of different selenium forms vary greatly. By simultaneously measuring multiple different selenium forms, the actual nutritional value and potential health benefits of selenium-enriched organisms can be more accurately assessed. The health benefits cannot be reliably predicted based solely on the total selenium content.

[0049] Furthermore, in some embodiments, in step S2, the buffer is Tris-HCl, the concentration of the buffer is 75 mmol, and the pH value of the buffer is 7.5.

[0050] In the technical solution of this application embodiment, the buffer can stabilize the pH value of the mobile phase, ensuring the consistency of the interaction between the solute and the stationary phase during the separation process. Furthermore, it can improve peak shape, increase resolution and selectivity, and protect the column from pH damage.

[0051] Furthermore, in some embodiments, step S1, the pulverization includes the following steps: The plant-based selenium-enriched food sample was pulverized in a high-speed pulverizer at 30,000 r / min for 3 minutes, passed through a 200-mesh sieve, and the sieved sample was collected.

[0052] Furthermore, in some embodiments, in step S1, the plant-based selenium-enriched food is selenium-enriched rapeseed, selenium-enriched tea, selenium-enriched rice, or selenium-enriched shepherd's purse powder.

[0053] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0054] Example 1 This embodiment provides a method for simultaneously determining nine selenium forms in plant-based selenium-enriched foods, specifically including the following steps: (1) Take a sample of selenium-enriched tea leaves, pulverize it for 3 minutes at 30,000 r / min using a high-speed pulverizer, pass it through a 200-mesh sieve, and take the sample that passes through the sieve to obtain selenium-enriched powder.

[0055] (2) Take 0.5g of the above selenium-enriched powder and place it in a centrifuge tube. Add 10mL of 1mol / L Tris-HCl buffer with a pH of 7.5 to the centrifuge tube. Then add 15.0mg of lysin and streptomycin E respectively. After sonicating at 37℃ with a power of 600W and a frequency of 40KHz for 40min, centrifuge at a speed of 10000r / min for 15min. Take the supernatant and filter it through a 0.22μm aqueous filter membrane to obtain the test solution.

[0056] (3) The above-mentioned test solution was subjected to qualitative and quantitative analysis using high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) to determine the selenium speciation in the test solution. Among them, the HPLC-ICP-MS instrument used was the Agilent 8800 oxygen dynamic reaction IC-MS instrument.

[0057] The chromatographic conditions are as follows: Mobile phase A: 15 mmol / L citric acid + 5 mmol / L sodium hexanesulfonate solution + methanol, wherein the pH of mobile phase A is 3.0, and the volume fraction of methanol in mobile phase A is 1%; Mobile phase B: 15 mmol / L citric acid + 5 mmol / L sodium hexanesulfonate solution; the pH of mobile phase A is 7.2; and the volume fraction of methanol in mobile phase B is 1%. Chromatographic column: Aglient ZORBAX SB-Aq reversed-phase chromatography column (250 mm × 4.6 mm, 5 μm); Column temperature: 20℃; Elution method: gradient elution; The parameters of the gradient elution are shown in Table 1: Table 1 Gradient elution conditions Mass spectrometry conditions are: Radio frequency power: 1550W; sampling depth: 8.0mm; nebulizer flow rate: 1.03L / min; peristaltic pump speed: 0.3rps; acquisition mode: O2; nebulizer temperature: 2℃; integration time: 0.5s; acquisition time: 540s; reaction gas: oxygen; reaction gas flow rate: 25%; mass numbers of Q1 and Q2: 78 and 94, respectively.

[0058] The standard chromatogram of selenium speciation in the test solution obtained in this embodiment is as follows: Figure 1 As shown.

[0059] Depend on Figure 1 It can be seen that this method can simultaneously extract and measure five selenium forms present in selenium-enriched tea leaves.

[0060] Preparation of standard solution: Take selenoethionine (SeEt), selenourea (SeUr), selenomethionine (SeMet), selenolanine dihydrochloride (3,3'-Selenobisalanine), and methylseleninic acid standards into the same 10mL volumetric flask, dissolve and dilute with ultrapure water to prepare a standard stock solution with a concentration of 1.0mg / L (the theoretical concentration of each selenium standard, expressed as Se). Then add methyl-selenocysteine ​​standard solution (MeSeCys), selenocysteine ​​standard solution (SeCys2), and selenite standard solution (SeO3). 2- Selenate standard solution (SeO4) 2- The selenium standard working solutions were diluted with ultrapure water to concentrations of 0 μg / L, 1 μg / L, 5 μg / L, 10 μg / L, 25 μg / L, 50 μg / L, and 100 μg / L.

[0061] To verify the accuracy and repeatability of the method, a spike recovery test was performed on blank samples.

[0062] Three levels of selenium standard working solutions (0.2 mg / kg, 0.4 mg / kg, and 0.8 mg / kg for each selenium form) were added to a blank sample matrix (selenium-free plant-based food). Subsequent operations were then performed to obtain the matrix-spiked solutions. These solutions were then tested under the conditions described in Example 1, with each level measured six times in parallel. The recoveries and relative standard deviations were calculated. The test results and data processing results are shown below. Figure 2 Tables 2 and 3.

[0063] Chromatogram of selenium speciation in matrix-spiked solution as shown in the figure. Figure 2 As shown.

[0064] Depend on Figure 2 It can be seen that the method of this application can effectively measure nine selenium forms simultaneously.

[0065] The results of the spiked recovery tests for the nine selenium forms are shown in Table 2.

[0066] Table 2. Spiking recoveries and precision of nine selenium forms As shown in Table 2, the recoveries of the nine selenium compounds at the three-level spiked levels ranged from 85.21% to 99.75%, and the relative standard deviations (n=6) ranged from 1.01% to 4.53%, indicating that the detection method has high sensitivity and good precision.

[0067] Table 3. Test results of selenium standard series working solutions, linear regression equation, correlation coefficient and detection limit. As can be seen from Table 3, the detection method in this application has a very good linear relationship.

[0068] An exploration of extraction methods for selenium speciation.

[0069] The effects of different proteases (proteinase K + streptinase (K + E), proteinase K + lyase (K + B), and streptinase + lyase (E + B)) on the extraction efficiency of nine selenium speciation compounds were investigated, and the results are as follows: Figure 3 As shown.

[0070] Figure 3 The results showed a significant statistical difference in extraction efficiency among different proteases (p<0.05), which was directly related to the broad-spectrum cleavage characteristics of the proteases and their degradation efficiency against the target selenium-containing proteins. The experimental results indicated that, among the tested combined enzyme systems, the E+B dual-enzyme combination, consisting of a breakdown enzyme and streptomycin, exhibited the best overall extraction efficiency. This combination was particularly effective in extracting organic selenium compounds such as methylselenocysteine ​​and selenomethionine. This may be due to the specific disruption of the polysaccharide structure of plant cell walls by the breakdown enzyme, effectively promoting the release of intracellular substances; simultaneously, streptomycin, with its broad-spectrum proteolytic activity, deeply degrades the released selenium-containing proteins, thereby efficiently converting bound selenium into soluble small-molecule selenium compounds. Other dual-enzyme combinations, such as K+B (protease K + breakdown enzyme) and K+E (protease K + streptomycin), also showed some synergistic effects, but the K+B combination was more advantageous for extracting inorganic selenium compounds, while the overall extraction efficiency of the K+E combination was relatively low. The above results fully demonstrate that the choice of enzymatic hydrolysis system is highly correlated with the chemical properties of the target selenium compound. Based on a comprehensive consideration of extraction efficiency, target product specificity, and economic cost, the E+B dual-enzyme combination was ultimately determined and adopted as the optimal enzymatic hydrolysis scheme.

[0071] The effects of different amounts of protease (enzyme to sample mass ratio 1-9%) on the extraction efficiency of nine selenium speciation compounds were investigated, and the results are as follows: Figure 4 As shown.

[0072] Figure 4The results show that the extraction efficiency of selenium initially increases and then plateaus with increasing protease dosage. When the enzyme dosage increases from 1% to 5%, the extraction efficiency significantly improves, indicating that the active sites of the protease are not saturated at low enzyme dosages, and increasing the enzyme dosage can promote the binding of more substrates, thereby increasing the reaction rate. However, when the enzyme dosage exceeds 5%, the increase in extraction efficiency slows down and tends to stabilize, indicating that the active sites of the enzyme are close to saturation, and the substrate becomes the limiting factor. Based on cost-benefit analysis, 3-5% was ultimately selected as the optimal enzyme dosage.

[0073] The effects of different enzymatic hydrolysis times (20–60 min) on the extraction efficiency of nine selenium speciation compounds were investigated, and the results are as follows: Figure 5 As shown.

[0074] Figure 5 The results show that the extraction efficiency first increases and then decreases with increasing enzymatic hydrolysis time. Within the range of 20-60 min, as the hydrolysis time increases, the protease and substrate interact fully, and the release of selenium significantly increases. The optimal extraction efficiency is reached at 40 min. However, further extending the hydrolysis time to 60 min leads to a decrease in extraction efficiency. Therefore, 38-42 min is the optimal hydrolysis time for selenium extraction.

[0075] The separation efficiency of mobile phases at different pH values ​​(3.0, 3.5, 4.5, 5.5, 6.5, 7.2) for nine selenium-containing compounds was investigated, and the results are as follows: Figure 6 As shown.

[0076] Figure 6 The results show that the retention time of SeO32- decreased monotonically from 2.501 min to 2.266 min with increasing pH (2.7~7.2), which is consistent with the change from HSeO32-. - (Low pH) to SeO3 2- (High pH) conversion is involved; while SeO4 2- The retention time remained stable at pH > 5.2, because it always retained the form of SeO4. 2- SeO4 exists in its natural form, and its charge distribution is unaffected by pH. The chromatographic behavior of organoselenium forms exhibits a more complex pH dependence; SeEt, Selenobisalanine, MeSeCys, and SeMet generally show a decreasing retention time with increasing pH. Furthermore, SeO4 can be obtained within the pH range of 2.7–3.5. 2- With SeO3 2-Baseline separation was achieved, but SeUr overlapped with MeSeCys. When the pH was increased to 5.2, methylseleninic acid, Selenobisalanine, and SeCys2 co-eluted. At pH 6.5, the separation of the five organoselenium forms significantly decreased, while the elution times of SeMet and SeEt were advanced to within 6 min. This indicates that pH modulates the interaction strength of selenium forms in the chromatographic system by altering their charge state, inducing molecular conformational changes, and affecting compound stability. This application achieved baseline separation of nine selenium forms using alternating mobile phase A (pH 3.0) and mobile phase B (pH 7.2).

[0077] The effects of methanol volume fraction (0%, 1%, 2%) in the mobile phase on the elution efficiency, retention time, and ICP-MS signal intensity of nine selenium speciation compounds were investigated, and the results are as follows: Figure 7 As shown.

[0078] Figure 7 The results show that when the methanol ratio increases from 0% to 1%, the peak intensity of each selenium speciation significantly increases. This is attributed to the fact that low-concentration methanol, as a carbon source, enhances the ionization efficiency of selenium in ICP-MS plasma (carbon effect). When the methanol ratio is >1%, the polarity of the mobile phase decreases, and the retention time of organic selenium speciation (such as SeCys2, SeMet, and SeEt) is significantly shortened due to the weakened hydrophobic interaction with the stationary phase, while the retention time of inorganic selenium speciation (SeO4) is significantly reduced. 2- SeO3 2- The retention times of SeUr and SeCys2 were largely unaffected. However, when the methanol ratio increased to 2%, the retention times of SeUr and SeCys2 became similar, leading to some peak overlap. Simultaneously, a high methanol ratio increased the organic loading of the ICP-MS plasma, potentially causing signal fluctuations. This indicates that a methanol addition ratio of 1% in the mobile phase ensured good separation and signal enhancement of each selenium form while avoiding peak overlap and signal instability issues caused by a high methanol ratio.

[0079] Using oxygen as a collision reactor, the effects of Se at a mass concentration of 10 ppb were investigated. 4+ The signal and background intensities of the standard and blank solutions under different oxygen contents (5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%) are shown in the figure. Figure 8 As shown.

[0080] Figure 8 The results show that when the gas volume fraction in the collision reaction cell is 25%, the blank intensity is relatively stable, the oxygen signal intensity is the best, and the BEC value is the lowest.

[0081] In summary, this application provides a method for simultaneously determining nine selenium forms in plant-based selenium-enriched foods. By optimizing the enzymatic hydrolysis conditions and the parameters of the chromatography-mass spectrometry (GC-MS) coupling, efficient extraction, accurate separation, and high-sensitivity detection of selenium forms are achieved. This method can simultaneously determine nine selenium forms: selenocysteine, methylselenocysteine, selenomethionine, selenoethionine, selenomethionine, selenolanethionine, selenate, selenite, and selenourea. The combination, dosage, and processing time of specific proteases can achieve efficient selenium extraction. These proteases exhibit good synergistic effects, optimizing the enzymatic hydrolysis system, avoiding competitive inhibition between enzymes, and improving extraction efficiency and cost-effectiveness. Specific mobile phase pH values ​​can alter the charged state of selenium speciation, induce molecular conformational changes, and affect compound stability, ultimately regulating their interaction strength in the chromatographic system. This ensures the retention behavior and high resolution of different selenium speciations on the chromatographic column, achieving baseline separation of multiple selenium speciations. By setting a specific methanol concentration in the eluent, good elution efficiency and signal enhancement for each selenium speciation are ensured, while avoiding peak overlap and signal instability caused by high methanol ratios. Combined with gradient elution, this enables simultaneous and efficient separation of different selenium speciations and efficient and stable detection of multiple selenium speciations.

[0082] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for simultaneously determining nine selenium species in plant selenium-rich food, characterized in that, The method comprises the following steps: S1. crushing a plant selenium-rich food sample to obtain a selenium-rich sample powder; S2. dissolving the selenium-rich sample powder in a buffer, adding a protease, ultrasonic enzymolysis, and then centrifugal separation to obtain a test solution; S3. determining nine selenium forms in the test solution by using high performance liquid chromatography-inductively coupled plasma mass spectrometry.

2. The method for simultaneous determination of nine selenium species in plant selenium-rich food according to claim 1, characterized in that, In step S2, the protease is lysozyme and pronase.

3. The method for simultaneous determination of nine selenium species in plant selenium-rich food according to claim 1, characterized in that, In step S2, the mass of the protease accounts for 3-5% of the selenium-rich sample powder.

4. The method for simultaneous determination of nine selenium species in plant selenium-rich food according to claim 1, characterized in that, In step S3, the conditions of the high performance liquid chromatography in the high performance liquid chromatography-inductively coupled plasma mass spectrometry are as follows: mobile phase A: 15 mmol / L citric acid + 5 mmol / L sodium hexanesulfonate solution + methanol, the pH value of the mobile phase A is 3.0, and the volume fraction of methanol in the mobile phase A is 1%; mobile phase B: 15 mmol / L citric acid + 5 mmol / L sodium hexanesulfonate solution, the pH value of the mobile phase A is 7.2, and the volume fraction of methanol in the mobile phase B is 1%; chromatographic column: Aglient ZORBAX SB-Aq reverse-phase chromatographic analysis column (250 mm x 4.6 mm, 5 μm); column temperature: 20℃; elution mode: gradient elution.

5. The method for simultaneous determination of nine selenium species in plant selenium-rich food according to claim 3, characterized in that, The parameters of the gradient elution are shown in the following table: 。 6. The method for simultaneous determination of nine selenium species in plant selenium-rich food according to claim 1, characterized in that, In step S2, the conditions of the inductively coupled plasma mass spectrometry in the high performance liquid chromatography-inductively coupled plasma mass spectrometry are as follows: power of radio frequency: 1550 W; sampling depth: 8.0 mm; flow rate of atomizer: 1.03 L / min; rotation speed of peristaltic pump: 0.3 rps; mode of collection: O2; temperature of atomization chamber: 2℃; integration time: 0.5 s; collection time: 540 s; reaction gas: oxygen; flow rate of reaction gas: 25%; mass numbers of Q1 and Q2: 78 and 94.

7. The method for simultaneous determination of nine selenium species in plant selenium-rich food according to claim 1, characterized in that, In step S2, in the ultrasonic enzymolysis, the enzymolysis time is 38-42 min, the temperature is 37℃, the ultrasonic power is 600 W, and the ultrasonic frequency is 40 KHz.

8. The method for simultaneous determination of nine selenium species in plant selenium-rich food according to claim 1, characterized in that, In step S2, the centrifugal separation comprises the following steps: centrifuging at a centrifugal rotation speed of 10000 r / min for 15 min, taking the supernatant, and passing the supernatant through a water filter membrane with a pore size of 0.22 μm to obtain the test solution.

9. The method for simultaneous determination of nine selenium species in plant selenium-rich food according to claim 1, characterized in that, In step S3, the nine selenium forms include selenocystine, methylselenocysteine, selenomethionine, selenoethionine, methylselenious acid, selenohomocysteine dihydrochloride, selenate, selenite, and selenourea.

10. The method for simultaneous determination of nine selenium species in plant selenium-rich food according to claim 1, characterized in that, In step S2, the buffer is Tris-HCl, the concentration of the buffer is 75 mmol, and the pH value of the buffer is 7.5.

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