Method for extracting, separating, qualitatively and quantitatively analyzing different selenium forms in food materials

Abstract

The invention discloses an extraction separation and qualitative and quantitative analysis method for different selenium forms in food materials. The method comprises the following steps: (1) pretreating a food material sample to obtain sample powder; (2) dissolving the selenium powder in water, performing ultrasonic extraction, centrifuging, taking supernate, and filtering to obtain a water-soluble selenium extracting solution; (3) protease XIV, basic protease, 2-hydroxyethanethiol and water are added into the solid residues for ultrasonic extraction; centrifuging, taking supernate, and filtering to obtain a combined organic selenium extracting solution; (4) adding a sodium sulfite aqueous solution into the solid residues in the step (3), and carrying out oscillation extraction; centrifuging and taking supernate to obtain an element state selenium (Se0) extracting solution; and (5) performing qualitative and quantitative analysis on different selenium forms in the extracting solution by adopting high performance liquid chromatography-inductively coupled plasma mass spectrometry. According to the method, water-soluble selenium can be respectively measured in a step-by-step extraction mode, the content of organic selenium and the content of elemental selenium (Se0) are combined, the problem that selenocystine cannot stably exist is solved by adding 2-hydroxyethanethiol, and the method has the advantages of being high in extraction rate, low in quantitation limit, good in stability and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the extraction and detection method of different selenium forms in food materials, in particular to the extraction and detection method of different selenium forms in food, and belongs to the field of separation, extraction and detection of selenium forms. BACKGROUND

[0002] Selenium is an essential trace element for the human body and cannot be self-made, has the effects of anti-cancer, delaying aging, enhancing human immunity, etc. Selenium-rich plants are an important way for the human body to obtain selenium, and supplementing the selenium content in the human body through daily diet is currently recognized as the best way to supplement selenium. However, selenium is a typical dual-functional element, which is beneficial to the human body when the concentration is within a certain range, but when the concentration exceeds a narrow level, it will be harmful to the human body. Selenium exists in selenium-rich plants in the form of selenium, which is divided into two forms: organic selenium (selenium cysteine, selenium methionine, methyl selenocysteine) and inorganic selenium (selenium salt, sodium selenite). Both can be absorbed by the human body, but the absorption rates and toxicities of the two are different. Quantitative analysis and determination of selenium forms in selenium-rich food are crucial for scientific selenium supplementation.

[0003] Quantitative extraction and separation detection of selenium forms in selenium-rich food are two key steps for selenium form determination. The extraction of selenium forms in selenium-rich food directly affects the later detection, and the selection of extraction solvent and the optimization of extraction conditions are very critical. The extraction method of selenium forms in selenium-rich food has obvious specificity, and different plant species or different selenium forms often require different extraction solvents, extraction conditions and extraction methods. Therefore, a universal method for extracting and detecting different selenium forms in food is urgently needed for the analysis and determination of selenium forms in selenium-rich food. SUMMARY

[0004] The main purpose of the present application is to provide a method for extracting and detecting different selenium forms in food, which has the characteristics of high extraction rate, low quantitative limit and good stability, and solves the problems of instability of sodium selenite, selenocysteine and other selenium forms, high matrix effect and unstable determination results in the determination of selenium forms in food.

[0005] In order to achieve the above purpose, the main technical solutions adopted by the present application include: A method for extracting, separating and qualitatively and quantitatively analyzing different selenium forms in food materials, comprising: Step (1) pretreating the food material sample to be tested to obtain a sample powder; Step (2) dissolving the sample powder with water and then ultrasonically extracting, centrifuging to take the supernatant and filtering to obtain a water-soluble selenium extract; Step (3) adding protease XIV, alkaline protease, 2-hydroxyethyl mercaptan and water to the solid residue after centrifugation in step (2) to obtain a mixture; ultrasonically extracting the mixture; centrifuging the extract to take the supernatant and filtering to obtain a combined organic selenium extract; Step (4) adding sodium sulfite aqueous solution to the solid residue obtained after centrifugation in step (3) for oscillation extraction; centrifuging the extraction liquid to obtain Se 0 extraction liquid; Step (5) using high performance liquid chromatography-inductively coupled plasma mass spectrometry to qualitatively and quantitatively analyze selenium in the extraction liquid obtained in step (1)-step (3).

[0006] In a preferred embodiment of the present application, the food material to be tested can be any food material containing selenium, such as fruits, vegetables, grains, tea leaves or edible fungi, etc.; preferably tomatoes.

[0007] In a preferred embodiment of the present application, the pretreatment method in step (1) is preferably: freeze-drying the food material sample at -80℃ and grinding into powder under liquid nitrogen.

[0008] In a preferred embodiment of the present application, the ultrasonic extraction conditions in step (2) are preferably: 20-30℃, 90-300 W power ultrasonic extraction for 20-80 min; further preferably, the ultrasonic extraction conditions are: 25℃, 150 W or 180 W power ultrasonic extraction for 40 min.

[0009] In a preferred embodiment of the present application, the centrifugation conditions in step (2) are preferably: 0-4℃, 4000-12,000 r / min centrifugation for 5-20 min; further preferably, the centrifugation conditions are preferably: 4℃, 8000 r / min centrifugation for 10 min.

[0010] In a preferred embodiment of the present application, the amount of proteinase XIV added in step (3) is 1-30% of the total mass of the mixture, preferably 5-25%, and most preferably 20%; the amount of alkaline protease added in step (3) is 1-30% of the total mass of the mixture.

[0011] It has been found through experiments that by adding 2-hydroxyethyl mercaptan, SeCys2enzymatically released from the sample can be converted into stable selenium form peaks, thereby realizing accurate determination of SeCys2. However, the addition of 2-hydroxyethyl mercaptan can cause SeO3 2- rapid decomposition, and the recovery rate is reduced to 0%; therefore, the present application extracts water-soluble selenium forms first, and then extracts combined organic selenium by adding 2-hydroxyethyl mercaptan to the extraction liquid, thereby avoiding rapid decomposition of SeO3 2- .

[0012] In a preferred embodiment of the present invention, the volume ratio of water to 2-hydroxyethyl mercaptan in step (3) is preferably (5000-20000):(5-50), and most preferably 10000:20.

[0013] In a preferred embodiment of the present invention, the ultrasonic extraction conditions in step (3) are preferably: ultrasonic extraction at 20-30℃ and 90-300 W power for 20-80 min; more preferably, the ultrasonic extraction conditions are: ultrasonic extraction at 25℃ and 150 W or 180 W power for 40 min.

[0014] In a preferred embodiment of the present invention, the centrifugation conditions described in step (3) are... Preferably, the centrifugation conditions are: 0-4℃, 4000-12,000 r / min for 5-20 min; more preferably, the centrifugation conditions are: 4℃, 8000 r / min for 10 min.

[0015] In a preferred embodiment of the present invention, the filtration described in step (2) or step (3) is performed using a 0.22 μm filter membrane.

[0016] In a preferred embodiment of the present invention, the high-performance liquid chromatography (HPLC) conditions in step (5) of the HPLC-ICP-MS are as follows: mobile phase: weigh 960 mg citric acid and 470 mg sodium 1-hexanesulfonate, add water to 500 mL, adjust the pH to 4.7 with ammonia, add 20 mL methanol, and degas by sonication for 5 min; column: Thermo Fisher Scientific Hypersil GOLDTMAQ (4.6 × 250 mm, particle size 5 μm); flow rate: 0.8-1.0 mL / min, gradient elution; injection volume: 5-10 μL.

[0017] In a preferred embodiment of the present invention, the conditions for inductively coupled plasma mass spectrometry (ICP-MS) in step (5) are as follows: RF power: 1560 W; concentric nebulizer; peristaltic pump rate: 0.4 r / min; carrier gas flow rate: 1.05 L / min; sampling depth: 9 mm; high-helium collision cell; Ar flow rate: 15 mL / min; sampling mass number 78 Se, integration time: 0.5-1.0 s.

[0018] In a preferred embodiment of the present application, the method for qualitative analysis of different selenium forms in the extract obtained in steps (1)-(3) in step (5) is preferably as follows: chromatograms of standard working solutions of different selenium forms and sample solutions of the foodstuff to be tested are determined respectively, and the chromatographic retention time of each compound in the standard working solution and the sample solution of the foodstuff to be tested is recorded; when the chromatographic peak in the sample solution of the foodstuff to be tested is consistent with the chromatographic retention time of the five selenium forms in the standard solution, the corresponding compound in the sample is determined; wherein the five selenium forms are selenocystine (SeCys2), methyl selenocysteine (MeSeCys), selenomethionine (SeMet), selenite (SeO3 2- ), and selenate (SeO4 2- ).

[0019] In a preferred embodiment of the present application, the method for quantitative analysis of different selenium forms in the extract obtained in steps (1)-(3) in step (5) is preferably as follows: a standard curve of different selenium forms is plotted with the concentration μg / L as the abscissa and the response chromatographic peak area (CPS) as the ordinate, and the content of each selenium form is calculated through the standard curve according to the corresponding peak area in the chromatogram of the sample solution to be tested.

[0020] In the present application, the selenium forms in selenium-rich food are determined by stepwise extraction; further, by optimizing the extraction temperature, extraction time, and extraction power, and by adding 2-hydroxyethyl mercaptan, the problem of low recovery rate and unstable existence of selenocystine in the extraction process of selenium forms in food is solved; by using the matrix standard addition method, the problem of matrix effect in the determination of selenium forms is solved; by using high performance liquid chromatography-inductively coupled plasma mass spectrometry, the accurate qualitative and quantitative analysis of five selenium forms in food is realized; according to the quantitative analysis results of the analysis method, the linear range of the five selenium forms is 0.5-60.0 μg / L; the quantification limit of selenocystine, methyl-selenocysteine, selenomethionine, selenite, and selenate is 2.0 μg / L. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The chromatogram of the five selenium forms is shown, from left to right, as selenate, selenite, selenocystine, methyl selenocysteine, and selenomethionine.

[0022] Figure 2 The standard curve of selenate is shown, with the concentrations being 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 6.0 μg / L, 12.0 μg / L, 30.0 μg / L, and 60.0 μg / L.

[0023] Figure 3 The standard curve for selenite is 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 6.0 μg / L, 12.0 μg / L, 30.0 μg / L and 60.0 μg / L in turn.

[0024] Figure 4 The standard curve for selenocystine is 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 6.0 μg / L, 12.0 μg / L, 30.0 μg / L and 60.0 μg / L in turn.

[0025] Figure 5 The standard curve for methylselenocysteine is 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 6.0 μg / L, 12.0 μg / L, 30.0 μg / L and 60.0 μg / L in turn.

[0026] Figure 6 The standard curve for selenomethionine is 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 6.0 μg / L, 12.0 μg / L, 30.0 μg / L and 60.0 μg / L in turn.

[0027] Figure 7 The chromatogram of the selenium-rich tomato sample extracted in step (2) is shown, and the first peak from left to right is methylselenocysteine.

[0028] Figure 8 The chromatogram of the selenium-rich tomato sample extracted in step (3) is shown, and the first peak from right to left is selenomethionine.

[0029] Figure 9 The extraction rate of 5 selenium forms and total selenium by different enzyme combinations is shown.

[0030] Figure 10 The extraction rate of 5 selenium forms and total selenium by different enzyme dosages is shown.

[0031] Figure 11 The recovery rate of selenite under different ultrasonic power and extraction time is shown.

[0032] Figure 12 The recovery rate of selenite under different ultrasonic power and extraction time is shown.

[0033] Figure 13 The recovery rate of selenocystine under different ultrasonic power and extraction time is shown.

[0034] Figure 14 The recovery rate of methylselenocysteine under different ultrasonic power and extraction time is shown.

[0035] Figure 15 The graph shows the recovery rate of selenomethionine at different ultrasonic powers and extraction times.

[0036] Figure 16 The chromatogram of selenocysteine ​​converted into a stable selenium form by 2-hydroxyethylthiol, from left to right: selenate, selenite, selenocysteine, methylselenocysteine, converted selenocysteine, and selenomethionine.

[0037] Figure 17 The chromatogram of the conversion of selenite ions to elemental selenium by 2-hydroxyethylthiol, from left to right: selenate ion, methylselenocysteine, converted selenocysteine, and selenomethionine. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0039] Example 1: Establishment of methods for the separation, extraction, and qualitative and quantitative analysis of different selenium forms in food ingredients. 1. Preparation of sample solution 1.1 Sample Pretreatment The food sample (tomato) to be tested was cleaned, dried, freeze-dried at -80℃, and ground into powder under liquid nitrogen for later use.

[0040] 1.2 Separation and Extraction of Sample Solutions with Different Selenium Forms Step 1: Separation and extraction of water-soluble selenium: Weigh 0.1 g of the pretreated tomato sample into a 50 mL centrifuge tube, add 10 mL of water, and extract by ultrasonication at 25℃ and 150 W for 20 min. Centrifuge at 4℃ and 8000 r / min for 10 min. Transfer all the solution to a new 50 mL centrifuge tube, and filter the supernatant through a 0.22 μm filter membrane into a sample vial for later use.

[0041] Step 2: Separation and extraction of organic selenium: Add 10 mg of protease XIV, 10 mg of basic protease, 10 mL of water, and 20 μL of 2-hydroxyethyl mercaptan to the solid residue after step 1 extraction. Sonicate at 25℃ and 150 W for 80 min, centrifuge at 4℃ and 8000 r / min for 10 min, and filter the supernatant through a 0.22 μm filter membrane into a vial for later use.

[0042] Third step: 0 valence selenium separation and extraction: 20 mL 1 mol / L sodium sulfite aqueous solution was added to the solid residue of the second step, and extraction was carried out at 90 ℃ with 200 r / min oscillation for 1 h, 8000 r / min centrifugation for 10 min, and the supernatant was taken for standby.

[0043] 2 Preparation of standard solution Preparation of blank matrix solution I: 0.1 g of pretreated blank tomato sample was weighed in a 50 mL centrifuge tube, 10 mL of water was added, ultrasonic extraction was carried out at 25 ℃ with 150 W power for 20 min, 4 ℃, 8000 r / min centrifugation for 10 min, all the solution was transferred to a new 50 mL centrifuge tube, the supernatant was filtered through a 0.22 μm filter membrane into a sample vial, and standby.

[0044] Preparation of blank matrix solution II: 10 mg of protease XIV and 10 mg of alkaline protease were added to the solid residue after the extraction in the above step, 10 mL of water, 20 μL of 2-hydroxyethyl mercaptan were added, ultrasonic extraction was carried out at 25 ℃ with 150 W power for 80 min, 4 ℃, 8000 r / min centrifugation for 10 min, the supernatant was filtered through a 0.22 μm filter membrane into a sample vial, and standby.

[0045] Preparation of standard solution: appropriate amounts of selenocystine (SeCys2, 0.560 μmol / g), methyl selenocysteine (MeSeCys, 0.433 μmol / g), selenomethionine (SeMet, 0.499 μmol / g), selenite (SeO3 2- , 0.543 μmol / g), and selenate standard (SeO4 2- , 0.525 μmol / g) solution were accurately weighed, and the corresponding standard solution intermediate solution (calculated by selenium) with a concentration of 3 mg / L was prepared.

[0046] Preparation of series standard working solution I: the corresponding volume of the above standard solution was diluted with matrix blank solution I to constant volume, and the series mixed standard working solution was prepared by stepwise dilution, and the mixed selenium standard working solution had a concentration range of 0.5-60.0 μg / L.

[0047] Preparation of series standard working solution II: the corresponding volume of selenocystine, methyl selenocysteine solution, and selenomethionine standard solution was diluted with matrix blank solution II to constant volume, and the series mixed standard working solution was prepared by stepwise dilution, and the mixed selenium standard working solution had a concentration range of 0.5-60.0 μg / L.

[0048] 3 Analysis conditions of high performance liquid chromatography-inductively coupled plasma mass spectrometry 3.1 Liquid chromatography separation conditions Mobile phase: 960 mg citric acid, 470 mg 1-hexane sulfonic acid sodium, add water to 500 mL, adjust pH to 4.7 with ammonia water, add 20 mL of methanol, ultrasonic degassing for 5 min; column: Thermo Hypersil GOLD TMAQ (4.6 x 250 mm, particle size 5 μm). Flow rate: 0.8-1.0 mL / min, isocratic elution; injection volume: 5-10 μL.

[0049] 3.2 Inductively coupled plasma mass spectrometry instrument conditions Radio frequency power: 1560 W; concentric atomizer; peristaltic pump rate: 0.4 r / min; carrier gas flow: 1.05 L / min; sampling depth: 9 mm, high helium collision reaction cell; Ar flow: 15 mL / min, sampling mass number 78 Se, integration time: 0.5-1.0 s.

[0050] 4 Qualitative analysis Determine the standard working solution and the sample solution to be tested respectively, and record the chromatographic retention time of each compound in the standard working solution and the sample solution to be tested. When the chromatographic peaks in the sample solution to be tested are consistent with the chromatographic retention time of the five selenium forms in the standard solution, it can be determined that the corresponding compounds are detected in the sample to be tested.

[0051] 5 Quantitative analysis Determine the standard working solution according to the instrument reference conditions, take the concentration (μg / L) as the abscissa and the response chromatographic peak area (CPS) as the ordinate, draw the standard curve, and calculate the content of each selenium form through the standard curve by the peak area corresponding to the sample solution chromatogram.

[0052] The content of the target selenium form in the sample solution to be tested is quantified by the standard curve external standard method, and the calculation formula is as follows: X=(c×V) / (m×1000)×K; X is the content of each selenium form in the sample to be tested, mg / kg; c is the concentration of the selenium form determined in the sample solution to be tested, μg / L; V is the volume of the extraction liquid, mL; m is the sample mass, g; K is the dilution multiple; the calculation result is kept to three significant figures.

[0053] Figure 2 The standard curve for selenate is: Figure 3 The standard curve for selenite is: Figure 4 The standard curve for selenocystine is: Figure 5 The standard curve for methylseleno cysteine is: Figure 6The standard curve of selenomethionine. Figure 7 is a chromatogram of the selenium-enriched tomato sample extracted in step (2), and from left to right, the first peak is methylselenocysteine; Figure 8 is a chromatogram of the selenium-enriched tomato sample extracted in step (3), and from right to left, the first peak is selenomethionine.

[0054] According to the quantitative analysis results of the analysis method, the linear range of the five selenium forms is 0.5-60 μg / L; the quantification limits of selenocystine, methylselenocysteine solution, selenomethionine, selenite, and selenate are all 2.0 μg / L.

[0055] Test Example 2 Optimization test of extraction methods of different selenium forms in food 1 Selection of protease types The extraction effects of different protease types on selenium forms in tomatoes were investigated. According to the characteristics of plant tissues, five different combinations of protease XIV (protease XIV is derived from Streptomyces griseus and is purchased from Merck, which is a general protease), protease XIV + cellulase, protease XIV + amylase, protease XIV + lysozyme, and protease XIV + alkaline protease were investigated for their extraction effects on selenium forms in tomatoes.

[0056] To the solid residue after the first step of extraction, 10 mg of protease XIV and 10 mg of alkaline protease were added, 10 mL of water and 20 μL of 2-hydroxyethyl mercaptan were added, and ultrasonic extraction was performed at 25°C and 150 W power for 30-120 min. The supernatant was centrifuged at 8000 r / min and 4°C for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane into a sample vial for standby.

[0057] The test results are shown in Table 1, which show that when protease XIV and alkaline protease are combined, the extraction effects of the two main selenium forms, methylselenocysteine and selenomethionine, and total selenium are the best. Figure 9

[0058] 2 Selection of enzyme dosage Since the cost of protease XIV is relatively high, the effects of 5 mg (5%), 10 mg (10%), 15 mg (15%), 20 mg (20%), and 25 mg (25%) of protease XIV dosage on the extraction of selenium forms were investigated.

[0059] The test results are shown in Table 2, which show that when the dosage of protease XIV is 10 mg (10%), the extraction effects of the two main selenium forms, methylselenocysteine and selenomethionine, and total selenium are the best. Figure 10

[0060] 3 Selection and optimization of ultrasonic power and ultrasonic time ​​Extraction was performed using different ultrasonic powers and ultrasonic times, and the recovery rates of different selenium forms were as follows: Figures 11-15 As shown, at 25℃, the recoveries of selenate, selenomethionine, and methylselenomethionine within 20-80 min were all in the range of 90%-125% at extraction powers of 120W-300W. Selenite and selenocysteine ​​showed poor stability and were significantly affected by ultrasonic power and time under these reaction conditions; among them, selenocysteine ​​had the worst recovery rate, ranging from 10%-30%, with the best recovery rate observed at an ultrasonic power of 150W.

[0061] The recovery rate of sodium selenite decreased with increasing ultrasound time at different ultrasonic powers, dropping below 80% when the time increased from 40 min to 80 min. Comparatively, 150 W and 180 W ultrasonic powers showed better results than other powers.

[0062] The extraction rate data show that selenocysteine ​​was almost undetectable at 150 W, with methylselenocysteine ​​and selenomethionine being the main selenium forms. Selenate showed the best extraction rate at 25℃ and 40 min, selenite at 35℃ and 40 min, methylselenocysteine ​​at 15℃ and 40 min, and selenomethionine at 25℃ and 40 min. Considering both recovery rate and total selenium content, 25℃ and 40 min were ultimately determined to be the optimal extraction conditions.

[0063] 4. The role of 2-hydroxyethylthiol from Figure 13 The data show that selenocysteine ​​has very poor stability in the sample matrix, with recoveries ranging from 10.6% to 30.5% under different optimized conditions. Adding 2-hydroxyethylthiol to the sample matrix can convert selenocysteine ​​into a stable selenium form, gradually changing the chromatographic peak from 3.5 min to 6.0 min. The recoveries at three different concentration levels ranged from 104.3% to 111.8% (see Table 5). Figures 16-17 2-Hydroxyethanethiol was added to the sample, and after mixing, the selenite peak disappeared during instrumental analysis. Figures 16-17 It is speculated that an excess of 2-hydroxyethanethiol would reduce sodium selenite to zero-valent selenium. Therefore, a stepwise extraction method was adopted after comprehensive consideration.

[0064] Experimental Example 3: Methodological Validation 1. Investigation of sample matrix effect The sample matrix effect (ME) was investigated by adding a certain amount of selenium form standard solution to the sample extraction solution. The sample matrix effect = 100% x (counting intensity of sample addition concentration - counting intensity of sample background) / counting intensity of standard solution concentration. If the matrix effect is < 80% or > 120%, it is considered that the matrix effect of the sample is large, and the influence of the matrix effect needs to be eliminated. As can be seen from Table 1, the sample has obvious matrix effect, and the matrix matching standard solution is used.

[0065] Table 1 Matrix effect of sample

[0066] 2 Method linearity and detection limit Prepare series of mixed standard solutions I containing 5 selenium form components and series of mixed standard solutions II containing 3 selenium forms (refer to test example 1), under the optimized test conditions, sample in turn, repeat 2 times, take the peak area count as the vertical coordinate (y), and the mass concentration as the horizontal coordinate (x) to establish the standard curve, and the determination coefficient (R 2 ) in the linear range is greater than 0.997. To determine the detection limit (LOD) and the quantification limit (LOQ), the lowest concentration point (0.5 µg / L) in the linear calibration curve is repeatedly injected into the high performance liquid chromatography system for at least 11 times, and the LOD is calculated by the ratio of 3 times the standard deviation to the slope, to evaluate the sensitivity of the method.

[0067] The linear equation, linear correlation coefficient, linear range and detection limit of 5 selenium forms under water extraction conditions are shown in Table 2.

[0068] Table 2 Linear equation, linear correlation coefficient, linear range and detection limit results of 5 selenium forms

[0069] The linear equation, linear correlation coefficient, linear range and detection limit of 3 selenium forms under enzyme extraction conditions are shown in Table 3.

[0070] Table 3 Linear equation, linear correlation coefficient, linear range and detection limit results of 3 selenium forms

[0071] 3 Recovery rate and precision after adding Tomato is taken as a representative sample, 5 selenium form standard solutions with 3 different concentrations (see Table 4 for specific concentrations) are added respectively, each addition level is repeated for 6 times in two days, and the average recovery rate (R) and precision (RSD) are calculated.

[0072] Table 4 Recovery rate and precision test results of 5 selenium forms in water extraction

[0073] Table 5. Results of recovery and precision tests for the three selenium forms in enzyme extracts.

[0074] The experimental results are shown in Tables 4 and 5. The recoveries of each component of selenium form under different extraction methods ranged from 89.4% to 114.8%, with relative standard deviations of 0.9% to 9.2%, indicating that the detection method established in this invention has good repeatability and high accuracy.

Claims

1. A method for the extraction, separation, and qualitative and quantitative analysis of different selenium forms in food ingredients, characterized in that, include: Step (1) Pre-process the food sample to be tested to obtain sample powder; Step (2) Dissolve the sample powder in water and extract it by ultrasonication. Centrifuge the extract, take the supernatant and filter it to obtain a water-soluble selenium extract. Step (3) Add protease XIV, basic protease, 2-hydroxyethylthiol and water to the solid residue after centrifugation in step (2) to obtain a mixture; extract the mixture by ultrasonication; centrifuge the extract, take the supernatant and filter to obtain the bound organic selenium extract; Step (4) Add sodium sulfite aqueous solution to the solid residue obtained after centrifugation in step (3) and extract by shaking; Centrifugation of the extract and collection of the supernatant yields Se. 0 Extract; Step (5) uses high performance liquid chromatography-inductively coupled plasma mass spectrometry to perform qualitative and quantitative analysis on the different selenium forms in the extracts obtained in steps (1) to (4).

2. The extraction, separation, and qualitative / quantitative analysis method according to claim 1, characterized in that, The food to be tested can be any food containing selenium; preferably any one of fruits, vegetables, grains, tea or edible fungi; most preferably tomatoes. The pretreatment method described in step (1) is as follows: freeze-dry the food sample to be tested at -80℃ and grind it into powder under liquid nitrogen.

3. The extraction, separation, and qualitative / quantitative analysis method according to claim 1, characterized in that, The conditions for ultrasonic extraction in step (2) are: 20-30℃, 90-300 W power ultrasonic extraction for 20-80 min; preferably, the conditions for ultrasonic extraction are: 25℃, 150 W or 180 W power ultrasonic extraction for 40 min. The centrifugation conditions in step (2) are: 0-4℃, 4000-12,000 r / min for 5-20 min; preferably, the centrifugation conditions are: 4℃, 8000 r / min for 10 min.

4. The extraction, separation, and qualitative / quantitative analysis method according to claim 1, characterized in that, The amount of protease XIV added in step (3) is 1-30% of the total mass of the mixture, preferably 5-25%, and most preferably 20%; the amount of alkaline protease added in step (3) is 1-30% of the total mass of the mixture.

5. The extraction, separation, and qualitative / quantitative analysis method according to claim 1, characterized in that, According to the volume ratio, the volume ratio of water to 2-hydroxyethyl mercaptan in step (3) is (5000-20000):(5-50), preferably 10000:

20.

6. The extraction, separation, and qualitative / quantitative analysis method according to claim 1, characterized in that, The conditions for ultrasonic extraction in step (3) are: 20-30℃, 90-300 W power ultrasonic extraction for 20-80 min; preferably, the conditions for ultrasonic extraction are: 25℃, 150 W or 180 W power ultrasonic extraction for 40 min. The centrifugation conditions in step (3) are: 0-4℃, 4000-12,000 r / min for 5-20 min; preferably, the centrifugation conditions are: 4℃, 8000 r / min for 10 min. The filtration described in step (2) or step (3) is performed using a 0.22 μm filter membrane.

7. The extraction, separation, and qualitative / quantitative analysis method according to claim 1, characterized in that, The conditions for high performance liquid chromatography in step (5) are as follows: Preparation method of mobile phase: Weigh 960 mg citric acid and 470 mg sodium 1-hexanesulfonate, add water to 500 mL, adjust the pH value to 4.7 with ammonia, add 20 mL methanol, and degas by sonication for 5 min to obtain the mobile phase; Column: Thermo Fisher Scientific Hypersil GOLDTMAQ; Flow rate: 0.8-1.0 mL / min, gradient elution; Injection volume: 5-10 μL.

8. The extraction, separation, and qualitative / quantitative analysis method according to claim 1, characterized in that, The conditions for inductively coupled plasma mass spectrometry (ICP-MS) in step (5) are as follows: RF power: 1560 W; concentric nebulizer; peristaltic pump rate: 0.4 r / min; carrier gas flow rate: 1.05 L / min; sampling depth: 9 mm; high-helium collision cell; Ar flow rate: 15 mL / min; sampling mass number 78 Se, integration time: 0.5-1.0 s.

9. The extraction, separation, and qualitative / quantitative analysis method according to claim 1, characterized in that, In step (5), the method for qualitative analysis of different selenium forms in the extracts obtained in steps (1)-(3) using high performance liquid chromatography-inductively coupled plasma mass spectrometry is as follows: the chromatograms of the standard working solutions and the food sample solutions of different selenium forms are measured respectively, and the chromatographic retention times of each compound in the standard working solutions and the food sample solutions are recorded. When the chromatographic peak in the food sample solution is consistent with the chromatographic retention times of the five selenium forms in the standard solution, the corresponding compound is detected in the sample. The five selenium forms are selenocysteine, methylselenocysteine, selenomethionine, selenite, and selenate.

10. The extraction, separation, and qualitative / quantitative analysis method according to claim 1, characterized in that, In step (5), the method for quantitative analysis of different selenium forms in the extracts obtained in steps (1) to (3) using high performance liquid chromatography-inductively coupled plasma mass spectrometry is as follows: a standard curve of five selenium forms is plotted with concentration μg / L as the abscissa and response chromatographic peak area as the ordinate. The content of each selenium form is calculated by using the corresponding peak area in the chromatogram of the sample solution to be tested through the standard curve. The five selenium forms are selenocysteine, methyl-selenocysteine, selenomethionine, selenite, and selenate.

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