A method for detecting various arsenic forms in grains using pressurized microwave extraction

Abstract

The present invention discloses a method for pressurized microwave extraction-assisted detection of various arsenic forms in grains, comprising the following steps: (1) sample pretreatment: hydrochloric acid extraction in the sample, heating and pressurizing extraction, centrifugal filtration to obtain an extract; (2) preparation of standard samples: preparation of standard solutions of hydrochloric acid and arsenic; (3) detection: liquid chromatography-inductively coupled plasma-mass spectrometry is used to detect the extract; a dual-column removal technology is used, an AG7 chromatographic column is connected before the six-way valve, and another AG7 chromatographic column is connected between the metering pump and the quantitative loop. A new microwave extraction method is established, the energy consumption of extraction is reduced, and the extraction efficiency is greatly improved; the interference problem of arsenic determination in millet is solved by ICP-MS / MS, and the accurate determination of arsenic in millet is achieved.

Description

Technical Field

[0001] The invention relates to the field of detection, and in particular to a method for pressurized microwave extraction-assisted detection of multiple arsenic forms in grains. Background Art

[0002] Arsenic can be divided into inorganic arsenic and organic arsenic. Inorganic arsenic is trivalent arsenic and pentavalent arsenic. Organic arsenic mainly includes arsenobetaine, dimethylarsenic, arsenocholine, monomethylarsenic, carbarsine, nitrophenylarsenic acid, 4-hydroxyphenylarsenic acid, roxarsone, etc. Previous studies generally monitored the total arsenic in millet, which was limited to the study of the total arsenic content. However, few scholars studied the arsenic forms in millet, resulting in the neglect of the toxicological effects of the arsenic forms in millet. The detection of arsenic forms in millet is mainly divided into two steps: one step is the extraction of arsenic forms in millet, and the other step is the detection of arsenic forms in millet.

[0003] The main reagents used for the extraction of arsenic forms are organic reagents, inorganic reagents, water and enzymatic extraction. The main extraction methods are shaking-assisted extraction, heating-assisted extraction, ultrasound-assisted extraction and microwave-assisted extraction. Among them, shaking-assisted extraction, heating-assisted extraction and ultrasound-assisted extraction are more commonly used extraction methods. The extraction temperature of these three extraction methods is low, but the extraction time is very long, up to several days. Microwave-assisted extraction is another auxiliary extraction method. It has major disadvantages. First, it is very easy to cause mutual conversion between arsenic forms. Second, each sample is in a separate digestion tank. The digestion temperature is controlled by the highest temperature or average temperature control method. The separate extraction tank absorbs microwaves and reacts. The matrix and digestion tank of each sample are different, resulting in different temperatures in each digestion tank and different extraction efficiencies. Third, the extraction time is relatively long.

[0004] At present, the main means of arsenic form detection include graphite furnace atomic absorption, atomic fluorescence, liquid-inductively coupled plasma mass spectrometry, liquid-organic mass spectrometry and other instrumental methods. The detection of arsenic forms by graphite furnace atomic absorption requires the separation of forms during the pretreatment process, and the detection of each element form separately. When atomic fluorescence is used to detect arsenic forms, some organic arsenic does not have a fluorescent response and cannot be detected. HPLC-ICP-MS has become the optimal element form detection method. It can realize online separation of element forms and detect multiple element forms in the sample with one injection. Compared with other detection methods, it has the advantages of high sensitivity, low detection limit and good stability. The main factor affecting the accuracy of ICP-MS determination of arsenic is mass spectral interference, which mainly includes isobaric interference, polyatomic ion interference, oxide interference and doubly charged ion interference. At present, the main modes for eliminating interference are collision mode and reaction mode. Although collision mode can eliminate polyatomic ion interference and oxide interference, it is easy to reduce sensitivity and it is difficult to eliminate the interference of doubly charged ions. In reaction mode, the target element reacts with the reaction gas to generate new ions, realize mass number transfer, and measure the new mass number, which can effectively eliminate interference. However, the principle of interference mode elimination is to effectively eliminate the original interference and not introduce new interference. The disadvantage of reaction mode is that new reaction ions are generated after mass number transfer, and the new reaction ions may bring new mass spectrum interference, affecting the accuracy of measurement. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention establishes a new microwave extraction method, reduces the energy consumption of extraction, and greatly improves the extraction efficiency; solves the interference problem of arsenic determination in millet by ICP-MS / MS, and determines the optimal interference removal mode by comparing different analysis methods, so as to achieve accurate determination of arsenic in millet.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for pressurized microwave extraction-assisted detection of multiple arsenic forms in grains comprises the following steps:

[0008] (1) Sample pretreatment: extract the sample with hydrochloric acid, extract under elevated temperature and pressure, and obtain the extract by centrifugation and filtration;

[0009] (2) Preparation of standard samples: prepare standard solutions of hydrochloric acid and arsenic;

[0010] (3) Detection: The extract was detected by liquid chromatography-inductively coupled plasma-mass spectrometry. The dual-column removal technology was used to connect an AG7 chromatographic column before the six-way valve and another AG7 chromatographic column between the metering pump and the quantitative loop.

[0011] Furthermore, the specific steps of sample pretreatment are as follows: weigh 5 g of sample, add 5 mL of 0.2% hydrochloric acid, extract for 20 min, set the extraction temperature at 80 °C, and set the auxiliary extraction pressure at 25 bar. After the extraction tube cools to room temperature, centrifuge at 8000 r for 5 min, take the supernatant, and filter it using a 0.22 μm filter membrane.

[0012] Furthermore, the interference removal column is: AG7 chromatographic column, with a size of 2*50mm. The AG7 chromatographic column realizes the removal of two interferences, one is the removal of interference in the eluent and the pump head, and the second is the removal of the dissolution background in the six-way valve and the metering pump in the inert liquid phase.

[0013] Furthermore, the conditions of liquid chromatography were: flow rate 0.4 ml / min, injection volume 5 μL, and mobile phase elution conditions as shown in the table below.

[0014]

[0015] Furthermore, the parameters of the mass spectrometer are as follows: arsenic detection uses dual mass number detection, the first stage mass number is 75, the second stage mass number is 91, and the oxygen flow rate is 0.45 mL / min.

[0016] Furthermore, the arsenic forms are: arsenic acid, arsenite, betaine, dimethylarsenic, arsenocholine, monomethylarsenic, p-ureidophenylarsonic acid, nitrophenylarsonic acid, p-nitrophenylarsonic acid, and roxarsone.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The present invention establishes a new microwave extraction method, changes the control mode of the extraction temperature, uses external temperature control technology for all extraction tubes, places all extraction tanks in the same microwave absorption liquid, and uses absorption liquid temperature control to eliminate the disadvantage that a single temperature control leads to different digestion temperatures and affects the experimental results. In view of the disadvantage that microwave-assisted extraction easily leads to changes in the arsenic form, nitrogen is introduced to exclude the air inside for protection, inhibit the conversion of the arsenic form, and achieve the effect of extracting the occurrence form of arsenic in millet. At the same time, the introduction of nitrogen adds a relatively strong pressure to reduce the interaction between millet and the extractant, accelerate the extraction of arsenic forms by the extraction solvent, reduce the energy consumption of extraction, and greatly improve the extraction efficiency.

[0019] The dual-column removal technology was used, with AG7 connected before the six-way valve, and another AG7 connected between the metering pump and the quantitative loop. The blank injection and standard substance solution injection comparison method was used to determine the removal effect of the arsenic background. The experimental results showed that there were two background peaks in the blank injection. These two background peaks were enriched by two AG7s, and formed a retention time difference with arsenic acid (ASV) in the standard substance solution, which achieved the accurate determination of trace arsenic in the target. Therefore, our newly established arsenic background removal method can more widely, more effectively and more accurately remove the arsenic background in liquid chromatography and mobile phases of different materials and different injection methods. At the same time, it can be foreseen that this method can be applied to other elements in the process of online determination. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of arsenic interference removal under different modes;

[0021] Figure 2 This is a schematic diagram of arsenic interference removal in tandem mode;

[0022] Figure 3 Optimized for oxygen conditions;

[0023] Figure 4 It is the spectrum curve of the online arsenic background interference removal effect, where curve B: blank spectrum curve without removal column, curve C: spectrum curve of standard solution without removal column, curve D: blank spectrum curve of single removal column before valve, curve E: spectrum curve of standard solution of single removal column before valve, curve F: blank spectrum curve of single removal column after valve, curve G: spectrum curve of standard solution of single removal column after valve, curve H: blank spectrum curve of double removal column, and curve I: spectrum curve of standard solution of double removal column. DETAILED DESCRIPTION

[0024] The present invention is further described below by means of specific examples. The following description is only exemplary and does not limit the content thereof.

[0025] Example 1

[0026] 1.1 Instrument and working parameters

[0027] Instrument: Liquid chromatography-inductively coupled plasma mass spectrometry instrument (1260-8900)

[0028] AG7 Thermo Fisher: 2*50mm

[0029] Chromatographic conditions: flow rate 0.4 ml / min, injection volume 5 μL, mobile phase elution conditions see Table 1 below.

[0030] Table 1 shows the mobile phase elution conditions.

[0031]

[0032] Mass spectrometry parameters: Arsenic detection uses dual mass number detection, the first-stage mass number is 75, the second-stage mass number is 91, and the oxygen flow rate is 0.45 mL / min.

[0033] 1.2 Standard solution

[0034] Use 0.2% nitric acid as solvent to prepare 0.1μg / L, 0.5μg / L, 1μg / L, 5μg / L, 10μg / L, 20μg / L, 30μg / L, 40μg / L, and 50μg / L.

[0035] 1.3 Sample pretreatment

[0036] Weigh 0.5 g of rice standard material of GBW(E)100361 into a polytetrafluoroethylene extraction tube, add 5 mL of 0.2% hydrochloric acid, extract for 20 min, set the extraction temperature to 80 °C, and use the auxiliary extraction pressure of 25 bar to extract the arsenic form. After the extraction tube cools to room temperature, centrifuge at 8000 r for 5 min, take the supernatant, filter it with a 0.22 μm filter membrane, and store the filtered extract at 4 °C. At the same time, perform a blank test and measure it using HPLC-ICP-MS / MS.

[0037] 2. Results and Discussion

[0038] 2.1 Optimization of extraction

[0039] First, we used common extraction solvents such as water, methanol water (1:1), methanol water (3:1), acetonitrile water (1:1), acetonitrile water (3:1), hydrochloric acid 1% (mass fraction, the following hydrochloric acid percentages are all mass fractions), hydrochloric acid 0.5% (mass fraction), and hydrochloric acid 0.2% (mass fraction) to extract arsenic forms in food.

[0040] Table 2 is a comparison of the extraction efficiency of different extraction solvents

[0041]

[0042] In the preliminary test, water, methanol water in different proportions, acetonitrile water and hydrochloric acid were used to extract the arsenic forms in the standard substance. The sum of the determination results of AS3 and AS5 was taken as the result of inorganic arsenic extraction, and the sum of the results of all arsenic forms was taken as the result of total arsenic. The determination results were compared with the reference values ​​of inorganic arsenic and total arsenic of the standard substance, and the extraction efficiency of inorganic arsenic and total arsenic was investigated.

[0043] Weigh 0.5g of rice standard material of GBW(E)100361 into a polytetrafluoroethylene extraction tube, add 5mL of water, methanol water (1:1), methanol water (3:1), acetonitrile water (1:1), acetonitrile water (3:1), hydrochloric acid 1%, hydrochloric acid 0.5%, respectively, and extract the arsenic forms with an extraction time of 30min, an extraction temperature of 80℃, and an auxiliary extraction pressure of 25bar. The experimental results in Table 2 show that the extraction efficiency of water as the extractant is the lowest, the extraction rate of inorganic arsenic is 37.3%, the extraction rate of total arsenic is 40.7%, and the extraction rate of AS3 is significantly lower. The best extraction effect is achieved with hydrochloric acid as the extractant, the extraction rates of inorganic arsenic are 99.2% and 99.4%, and the extraction rates of total arsenic are 103.3% and 103.8%. From the experimental results of the extraction, hydrochloric acid is a suitable extractant. However, the extraction efficiency of methanol and acetonitrile as extractants was significantly higher, with the extraction rate of inorganic arsenic ranging from 156.4% to 204.8% and the extraction rate of total arsenic ranging from 130.3% to 170.7%. From the experimental results, the main reason for the high experimental results when methanol was used as an extractant was the high results of arsenobetaine (ASB) and arsenous acid (AS3), and the main reason for the high results when acetonitrile was used as an extractant was the high results of AS3. Moreover, as the concentrations of methanol and acetonitrile increased, the results increased accordingly. The retention time of these two arsenic forms is short. Because we used an ion column for arsenic form analysis, the retention time for organic reagents is short. Therefore, we suspect that the sensitization effect of organic reagents caused the high values ​​of these two arsenic forms. Therefore, we further investigated the effect of the extraction solvent on arsenic form compounds.

[0044] In order to investigate the effect of different extraction solvents on arsenic forms, 10.0 μg / L standard substance solutions were prepared using water, methanol water (1:1), methanol water (3:1), acetonitrile water (1:1), acetonitrile water (3:1), hydrochloric acid 1%, and hydrochloric acid 0.5%, respectively. Then, 5 mL of the above standard solutions were added to polytetrafluoroethylene extraction tubes, and the extraction time was 30 min, the extraction temperature was 80 ° C, and the auxiliary extraction pressure was 25 bar. The experiment of the change of arsenic form standard substance solution was carried out. The experimental results show that when water, hydrochloric acid 0.5% and hydrochloric acid 1% are used as solvents, all arsenic form determination values ​​do not change significantly. The extraction solvents of methanol and acetonitrile had obvious sensitization effect on the signals of some arsenic forms. When methanol was used as the extraction solvent, the measured values ​​of ASB, AS3 and DMA were 19.2μg / L, 13.3μg / L and 10.5μg / L when the concentration of methanol was (1:1). When the concentration of methanol was (3:1), the measured values ​​of ASB, AS3 and dimethylarsenic (DMA) were 19.2μg / L, 13.3μg / L and 10.8μg / L. Methanol solution had obvious sensitization effect on ASB, AS3 and DMA, and the sensitization effect on ASB was the most obvious, followed by AS3, and the sensitization effect on DMA was small. There was no obvious sensitization effect on other arsenic forms, and the sensitization effect became more obvious with the increase of methanol concentration. We believe that the sensitizing effect of organic solvents on different arsenic forms depends on the retention time of the arsenic form. The shorter the retention time, the more obvious the sensitizing effect. As the retention time of the arsenic form compound increases, the effect gradually decreases until there is no effect. However, the results of acetonitrile water are not like this.

[0045] When the concentration of acetonitrile water is (1:1), the measured values ​​of ASB, AS3 and DMA are 10.1μg / L, 21.9μg / L and 10.9μg / L. When the concentration of acetonitrile water is (3:1), the measured values ​​of ASB, AS3 and DMA are 12.4μg / L, 29.1μg / L and 11.6μg / L. From the results of acetonitrile solution as the extraction solvent, it can be seen that the three arsenic forms with the shortest retention time, ASB, AS3 and DMA, are affected by acetonitrile and have obvious sensitization effects, and the sensitization effect becomes more obvious with the increase of acetonitrile content. However, when acetonitrile is the extraction solvent, AS3 is the most obviously affected by the sensitization effect, followed by ASB and DMA, and other arsenic form compounds have no obvious effect. Therefore, the sensitization effect of arsenic form compounds by organic matter is not simply positively correlated with the retention time, but is related to the type of organic matter, and the intensity of sensitization varies with different organic solvents.

[0046] Therefore, according to the extraction efficiency of standard substances, organic reagents are the least suitable choice as extraction agents because they have obvious sensitization effects on some arsenic forms, while water has a low extraction efficiency and is not a suitable choice. Finally, hydrochloric acid was selected as the extraction solvent.

[0047] 2.1.1 Optimization and confirmation of extraction conditions

[0048] According to the extraction efficiency of the standard substance, hydrochloric acid was finally determined to be the most suitable extraction solvent, and the extraction temperature, hydrochloric acid concentration and extraction time were further optimized. The extracted solution was measured using HPLC-ICP-MS / MS, and the sum of the measurement results of AS3 and AS5 was taken as the result of inorganic arsenic extraction, and the sum of the results of all arsenic forms was taken as the result of total arsenic. The measurement results were compared with the reference values ​​of inorganic arsenic and total arsenic of the standard substance to determine the final extraction conditions.

[0049] 2.1.2 Optimization of extraction temperature

[0050] Weigh 0.5g GBW(E)100361 rice standard material into a polytetrafluoroethylene extraction tube, add 5mL hydrochloric acid 0.2%, set the extraction temperature to 60, 70, 80, 90, 100℃, the auxiliary extraction pressure is 25bar, the extraction time is 20min, and after the extraction tube cools to room temperature, centrifuge at 8000r for 5min, take the supernatant, filter it with a 0.22μm filter membrane, store the filtered extract at 4℃, and do a blank test at the same time. HPLC-ICP-MS / MS is used for determination. The experimental results show that when the extraction temperature is 60℃, the extraction rate of inorganic arsenic is 43.0%, and the extraction rate of total arsenic is 46.3%. The extraction temperature increases, and the extraction rate increases. When the extraction temperature is 80℃, the extraction rate of inorganic arsenic increases from 43.0% to 101.3%, and the extraction rate of total arsenic increases from 46.3% to 104.4%. The extraction temperature continues to increase, and the extraction rate does not change significantly. Therefore, the optimal extraction temperature was determined to be 80°C.

[0051] 2.1.3 Extraction solvent optimization

[0052] Take 0.5g of rice standard substance of GBW(E)100361 in a polytetrafluoroethylene extraction tube, add 5mL of 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.15%, 0.2%, 0.4%, and 0.6% hydrochloric acid respectively, the extraction temperature is 80℃, the auxiliary extraction pressure is 25bar, and the extraction time is 20min. After the extraction tube cools to room temperature, centrifuge at 8000r for 5min, take the supernatant, filter it with a 0.22μm filter membrane, store the filtered extract at 4℃, perform a blank test at the same time, and measure it using HPLC-ICP-MS / MS. The experimental results show that when the hydrochloric acid concentration is 0.01%, the recovery rate of inorganic arsenic is only 47.2%, and the extraction rate of total arsenic is 52.5%. As the acidity increases, the extraction rate also increases. When the acidity increases to 0.2%, the extraction rate of inorganic arsenic is 101.7%, and the extraction rate of total arsenic is 104.6%. The extraction rate of inorganic arsenic and total arsenic does not change significantly as the hydrochloric acid concentration in the extraction solution continues to increase. Therefore, the extraction solvent was finally determined to be 0.2% hydrochloric acid.

[0053] 2.1.4 Optimization of extraction time

[0054] Take 0.5g GBW(E)100361 rice standard material in a polytetrafluoroethylene extraction tube, add 5mL0.2% hydrochloric acid, the extraction temperature is 80℃, the auxiliary extraction pressure is 25bar, the extraction time is 5, 10, 20, 30min, respectively. After the extraction tube cools to room temperature, centrifuge at 8000r for 5min, take the supernatant, filter it with a 0.22μm filter membrane, store the filtered extract at 4℃, do a blank test at the same time, and use HPLC-ICP-MS / MS for determination. The experimental results show that when the extraction time is 5min, the recovery rate of inorganic arsenic can reach 92.0%, and the extraction rate of total arsenic is 95.1%. As time increases, the extraction rate also increases. When the extraction time is 20min, the extraction rate of inorganic arsenic is 100.8%, and the extraction rate of total arsenic is 104.1%. When the extraction time continues to increase, the extraction rate of inorganic arsenic and total arsenic has no significant change. Therefore, the final extraction time is determined to be 20min.

[0055] When optimizing the extraction time, we found that when the extraction time was 5 minutes, the extraction rate of inorganic arsenic could reach 92.0%, and the extraction rate of total arsenic could reach 95.1%. Therefore, we suspected that a better extraction rate could be achieved under the conditions of 0.2% acidity and 80°C. Therefore, we used the heating-assisted extraction method for comparative investigation.

[0056] Take 0.5g GBW(E)100361 rice standard material in a polytetrafluoroethylene extraction tube, add 5mL 0.2% hydrochloric acid, heat-assisted extraction temperature is 80℃, extraction time is 5 and 20min respectively, after the extraction tube cools to room temperature, centrifuge at 8000r for 5min, take the supernatant, filter with a 0.22μm filter membrane, store the filtered extract at 4℃, do a blank test at the same time, and use HPLC-ICP-MS / MS for determination. The experimental results show that when ordinary heating-assisted extraction is used, there is no auxiliary extraction pressure, and the extraction time is 5min, the extraction rate of inorganic arsenic is 37.4%, and the extraction rate of total arsenic is 49.1%. Both extraction rates are far lower than the method we established, and the extraction effect of inorganic arsenic is significantly lower than that of organic arsenic. When the extraction time is 20min, the extraction rate of inorganic arsenic is 74.2%, and the extraction rate of total arsenic is 79.3%. Under the same time, it is far lower than the extraction effect of the established method, and even much lower than the extraction effect of 5min.

[0057] The experimental results using different concentrations, different times, different temperatures, different extraction solvents and different extraction methods show that when conducting arsenic form extraction experiments, the extraction effect of inorganic arsenic is lower than that of organic arsenic, especially the extraction rate of trivalent arsenic is most affected by the extraction conditions. This is contrary to the general belief that inorganic arsenic has better solubility and the extraction efficiency should be higher. This may be related to the way inorganic arsenic is combined in the sample.

[0058] 2.2 Optimization of mass spectrometry conditions

[0059] 2.2.1 Optimization of analytical methods

[0060] The mass spectrum interference of ICP-MS is mainly isobaric interference, polyatomic ion interference, oxide interference and doubly charged ion interference. Common interferences for arsenic determination are shown in Table 3. Various elements will affect the determination of arsenic, so it is a challenging task to accurately determine arsenic in millet. At present, the interference removal modes for arsenic determination are mainly collision mode and reaction mode. We used Nogas, He collision mode, oxygen reaction mode and tandem mode to remove the interference. 75 The mass spectrometry interference and removal of As were investigated.

[0061] Table 3 shows the mass spectrometry interferences encountered during arsenic determination

[0062]

[0063] Nogas Mode

[0064] In order to investigate the effect of different elements on arsenic, standard solutions of As, Fe, K, Se, Cl, V, Ca, Ni, Nd, Sm, Zr, and Cu were selected by ICP-MS with mass-to-charge ratio ( m / z )75 for determination. The experimental results are shown in Figure 3 .

[0065] Nogas mode is to introduce the solution to be tested into ICP-MS without adding any collision gas or reaction gas in the collision / reaction cell. The main significance of Nogas is to verify whether the element will cause mass spectrometry interference to the arsenic determination. The experimental results clearly show that Fe, K, Se, Cl, V, Ca, Ni, Nd, Sm, Cu, Co and other elements have a large interference with the determination of As, affecting the accuracy of arsenic determination.

[0066] In He mode, the solution to be tested is introduced into ICP-MS, and He gas is introduced into the collision / reaction cell as the collision gas. The main principle of interference removal is that polyatomic interfering ions and oxide interfering ions have a relative 75 As has a larger collision cross section. After the He gas is introduced, it will collide with the single ion 75 Compared with As, polyatomic interfering ions and oxide interfering ions will lose more kinetic energy during the collision process of He gas. Setting an energy barrier after the collision cell can prevent polyatomic interfering ions and oxide interfering ions from entering the quadrupole, thereby removing the interference of polyatomic interfering ions and oxide interfering ions Fe, K, Se, Cl, V, Ca, Ni, Cu, Co and other elements on arsenic determination. The experimental results show that after selecting the collision mode, it can effectively eliminate the interference of Fe, K, Se, Cl, V, Ca, Ni, Br, Cu, Co and other elements on arsenic determination, and has a good interference elimination ability. However, it cannot remove 150 Sm++, 150 The two doubly charged ions Nd++ interfere with the determination of arsenic. At the same time, the sensitivity of arsenic is reduced by more than ten times. Therefore, the collision mode is not the most ideal interference removal mode.

[0067] Reaction mode: introduce the test solution into ICP-MS and introduce oxygen as reaction gas into the collision / reaction cell. The basic principle of the reaction mode to remove interference is to introduce O into the reaction cell. 2 , some ions in the ion beam will react with O 2 Whether the ions in the particle beam react with oxygen depends on whether the reaction is exothermic or endothermic. When the reaction is exothermic, the ions can react with oxygen. When the reaction is endothermic, a weak reaction or no reaction occurs. After the ions react with oxygen, the target ions can be converted into new reaction product ions. The reaction product ions can be obtained at a new mass-to-charge ratio ( m / z) is measured under the reaction mode, but the reaction mode requires that the newly generated ions should be at a new mass-to-charge ratio ( m / z ) There is no new interference. The reaction between arsenic and oxygen is exothermic. 75 As and 16 O reacts to generate new reactive ions. 91 ( 75 As+ 16 O). The experimental results show that the oxygen reaction mode is used for the determination of 91 ( 75 As+ 16 O) can effectively eliminate polyatomic ion interferences, oxide interferences, and doubly charged ions observed in Nogas and He reaction modes. 91 New interferences were observed at Zr, so the reaction mode was not an ideal interference removal mode.

[0068] In tandem mode, compared with the traditional quadrupole ICP-MS, the triple tandem ICP-MS adds a quadrupole before the collision / reaction cell. The particle beam first passes through the first quadrupole, which screens the ions in the particle beam, and then performs mass number transfer in the collision / reaction cell to generate new reaction ions. The second quadrupole should treat the newly generated ions at a new mass-to-charge ratio ( m / z ) is selected, and the new mass-to-charge ratio ( m / z ) for quantification. Figure 2 shown.

[0069] In this experiment, the first quadrupole mass-to-charge ratio ( m / z ) is set to 75, then the target ion 75 As and interfering ions 75 (38Ar+37Cl), 75 (16O+59Co), 75 (40Ar+35Cl), 75 (16O+1H+61Ni), 75 (12C+63Cu), 75 (16O+1H+58Fe), 75 (14N+61Ni), 75 ( 23 Na+ 1 H+ 51 V), 75 (1H+74Se), 75 (17O+2H+56Fe), 75 (36Ar+39K), 75 (16O+16O+43Ca), 75 ( 150 Sm ++ ),75 ( 150 Nd ++ ) At the same time, oxygen is introduced into the reaction cell through the first quadrupole. 75 As and oxygen react in the collision cell to generate new reactive ions. 91 ( 75 AS+ 16 O), the second quadrupole sets the mass-to-charge ratio ( m / z ) is 91, then only 91 ( 75 AS+ 16 O). The experimental results are shown in Figure 2 .

[0070] The experimental results show that the cascade mode can remove the problems that the collision mode cannot solve. 75 ( 150 Sm ++ ), 75 ( 150 Nd ++ ) The shortcomings of the two double-charge interference removal can also be solved in the reaction mode 91 Zr 91 ( 75 AS+ 16 O) interference, because the first quadrupole has already adjusted the mass-to-charge ratio ( m / z ) is 75 for screening, 91 Zr will not enter the second quadrupole for new reagent ions 91 ( 75 AS+ 16 O) will cause interference, and the tandem mode has a higher sensitivity. Therefore, the tandem mode is the best method for arsenic determination.

[0071] 2.2.2 Optimization of reaction gas flow rate

[0072] In order to obtain the best interference removal effect, we optimized the oxygen flow rate. The oxygen flow rate was optimized to the lowest background equivalent concentration, with low background and high sensitivity as the principle, that is, the background value of the blank solution was low and the signal value in the standard solution was the highest, to obtain the optimal oxygen flow rate. The experimental results are shown below. Figure 3 The experimental results show that the signal values ​​of the blank and standard solutions both show a trend of first rising and then falling as the oxygen flow rate increases. At the beginning, as the oxygen flow rate increases, the reaction between oxygen and arsenic generates an increase in new ions, reaching a maximum at an oxygen flow rate of 0.45mL / min. As the oxygen flow rate continues to increase, the response value decreases. This is because when the oxygen flow rate increases, the collision gas will play a role, resulting in a decrease in the signal value. Taking into account the blank response value and the standard solution signal value, it is finally determined that the oxygen flow rate of 0.45mL / min is the optimal oxygen flow rate.

[0073] 2.2.3 Investigation of chromatographic conditions

[0074] Arsenic is a common metalloid and widely exists in the environment. Therefore, it may exist in the mobile phase and the internal facilities of the instrument. The arsenic speciation we established can analyze ten different arsenic forms at the same time, including both inorganic and organic forms, and the chemical structures and chemical properties of different forms are quite different. In order to achieve the simultaneous separation of inorganic and organic forms, we use the gradient elution method for analysis. For ASV, under the condition of low concentration eluent, ASV and the stationary phase of the separation column are combined. As the concentration of the eluent increases, ASV and the stationary phase of the chromatographic column are exchanged and eluted. In order to maintain the stability of chromatographic separation, the last section of the elution procedure is generally a low concentration equilibrium. Therefore, if the eluent or the internal facilities of the instrument contain ASV, the ASV contained in the eluent and the internal facilities of the instrument will accumulate on the chromatographic column at the beginning of the elution procedure under low concentration conditions, and finally be eluted with the target ASV instrument in the analysis sample, and the amount accumulated each time will be different. The method of removing arsenic background in the mobile phase in the prior art cannot solve the problem of arsenic background in the mobile phase.

[0075] We systematically investigated the ASV problems caused by the instrument and the background of ASV, and established a new background removal method. We performed arsenic speciation analysis according to the established arsenic speciation analysis method. When performing blank analysis, we found a chromatographic peak at the retention time of ASV, so we adopted the analysis method without adding solution. The experimental results are shown in Figure 4 In the middle curve B, a chromatographic peak is found. This chromatographic peak must be the blank caused by the mobile phase or the instrument itself. In order to verify whether it will affect the target arsenic form, we use the same analysis method to analyze a variety of arsenic form standard substances. The chromatographic curve results are shown in Figure 4 In curve C, it is found that the chromatographic peak that appears in the blank injection has the same retention time as ASV, so it is suspected that the interfering substance is ASV, ASV contained in the new mobile phase or instrument. In order to remove this arsenic background, we use the pre-valve column technology to remove interference. An AG7 chromatographic column is connected in front of the six-way valve. Theoretically, if only the eluent contains ASV, in the initial stage of the elution procedure, the arsenic background in the mobile phase will be enriched on the chromatographic column AG7. As the concentration of the eluent increases, ASV will be washed down, and the ASV in the eluent and the ASV in the target on the analytical column will form a retention time difference, resulting in two chromatographic peaks. The effect and results of background removal were determined by comparing blank injection and standard substance injection. The experimental results show that two chromatographic peaks appeared during blank injection ( Figure 4 Curve D in the middle), and the chromatographic curve of the standard solution ( Figure 4For comparison, one of the chromatographic peaks can be separated from the ASV of the standard solution, but the retention time of the other peak is the same as that of the ASV in the standard solution.

[0076] Connecting AG7 in front of the six-way valve did not solve the problem of arsenic background, because two chromatographic peaks appeared during blank injection, so we suspected that one was in the mobile phase and the other was caused by the instrument itself. Because this experiment used a bio-inert liquid phase, this instrument has a built-in coating, which can be better resistant to acids and alkalis, and there will be no metal in the overall pipeline, which has greater advantages for metal element morphology analysis. However, this new instrument brings new problems. In order to solve this problem, an AG7 is added between the metering pump and the quantitative loop to try to eliminate the problems of mobile phase and instrument dissolution together. The method of blank injection and standard substance solution injection comparison was used to determine the removal effect of arsenic background. The experimental results show that a large chromatographic peak and a very small chromatographic peak appear in the blank injection ( Figure 4 In the middle curve F), the larger chromatographic peak and the chromatographic result of the standard substance solution ( Figure 4 The chromatographic peak retention time of ASV in the middle curve G) is different, which achieves the separation of chromatographic peaks. The retention time of the smaller chromatographic peak is consistent with the chromatographic peak retention time of ASV in the standard substance, but still no satisfactory experimental results are obtained.

[0077] After analyzing the entire injection process, it was found that the liquid chromatograph uses valve switching technology during the injection process. During the sample analysis process, the mobile phase passes through the six-way valve-metering pump-quantitative loop-injection needle-chromatographic column in sequence. However, during the injection process, the mobile phase does not pass through the metering pump, quantitative loop and injection needle. The mobile phase passes through the six-way valve directly into the chromatographic column. Because the mobile phase contains ASV background, the arsenic background in the mobile phase will be enriched on the chromatographic column during the injection process, so a smaller chromatographic peak background will be caused during the switching process. In order to completely remove this interference, the dual-column removal technology is used. AG7 is connected in front of the six-way valve, and another AG7 is connected between the metering pump and the quantitative loop. In this way, during the injection process, the initial eluent concentration is low. At this time, the arsenic in the mobile phase will be adsorbed by the AG7 in front of the six-way valve, and the mobile phase that flows through the six-way valve and directly enters the chromatographic column is a "purified" eluent. The removal effect of the dual-column arsenic background was determined by comparing the blank injection and the standard substance solution injection. The experimental results show that one or two background peaks appear in the blank injection ( Figure 4 The two background peaks are enriched by two AG7s, which are different from the chromatographic results of the standard substance solution ( Figure 4By comparing the two background chromatographic peaks of the blank injection with the ASV chromatographic results of the standard substance solution, it was found that the retention time difference was achieved, which enabled the accurate determination of trace arsenic in the target. Therefore, our newly established arsenic background removal method can more widely, effectively and accurately remove the arsenic background in liquid chromatography and mobile phases of different materials and different injection methods. At the same time, it can be foreseen that this method can be applied to the online determination of other elements.

[0078] 3. Actual sample testing

[0079] The method of this study was used to measure millet purchased randomly in the market, and the results are shown in Table 4.

[0080] Table 4 shows the actual measurement results of millet samples

[0081]

[0082] After method confirmation, the method we established has the advantages of high sensitivity, high accuracy and good selectivity, and is suitable for the detection of millet samples. We applied the established method to the detection of millet samples. We determined 10 different arsenic forms in 12 batches of millet samples, and detected 4 arsenic forms in millet samples, DMA, MMA, AS3 and ASV. Among them, DMA, AS3 and ASV were detected in all 12 batches of samples, and MMA was detected in only one batch of samples.

[0083] The content of total arsenic and inorganic arsenic in different millet samples varies greatly. The content of total arsenic ranges from 10.41μg / kg to 118.15μg / kg, with an average of 25.91μg / kg, and the content of inorganic arsenic ranges from 9.45μg / kg to 116.76μg / kg, with an average of 24.55μg / kg. The content of AS3 ranges from 6.61μg / kg to 53.77μg / kg, with an average of 16.91μg / kg, the content of ASV ranges from 1.32μg / kg to 62.98μg / kg, with an average of 7.64μg / kg, and the content of DMA ranges from 0.459μg / kg to 2.95μg / kg, with an average of 1.25μg / kg. From the experimental results, the various arsenic forms, total arsenic and inorganic arsenic in millet vary greatly, but we have found some interesting rules and phenomena.

[0084] The total arsenic content in millet varies greatly, with the highest value being 11 times the lowest value, and the overall difference is large. However, after careful analysis of the data, we found that we analyzed 12 batches of millet samples, among which the total arsenic content in 11 batches of samples ranged from 10.41μg / kg to 31.21μg / kg, but the total arsenic content of sample No. 12 was higher at 118.15μg / kg. In other words, the overall content of total arsenic in millet is not high, but the content of sample No. 12 is higher, which may be related to the specific growth environment and processing technology.

[0085] The inorganic arsenic content of millet is 88.1%~98.8% of the total arsenic, with an average of 92.6%, which shows that the main form of arsenic in millet is inorganic arsenic, and the linear correlation coefficient between inorganic arsenic and total arsenic is 0.9995. The content pattern of inorganic arsenic is similar to that of total arsenic. The inorganic arsenic content of 11 batches of samples is between 9.45μg / kg and 28.91μg / kg, but the inorganic arsenic content of sample No. 12 is 116.76μg / kg, and the inorganic arsenic content of this batch of samples is significantly higher than that of other batches of samples.

[0086] Further analysis of the arsenic forms in the 12 batches of millet samples showed that the content of AS3 in the first 11 batches of samples accounted for 60.9%~88.9% of the total arsenic, with an average of 77.1%. The content of AS3 accounted for 66.9%~92.9% of inorganic arsenic, with an average of 80.6%. From the results, it can be seen that AS3 is the main arsenic form in the first 11 batches of samples, and the content of AS3 is higher than ASV. However, sample No. 12 showed a different pattern from the first 11 batches of samples. AS3 accounted for 45.5% of the total arsenic, inorganic arsenic accounted for 46.1%, and ASV became the main arsenic form. Sample No. 12 showed significantly different phenomena from other samples in terms of total arsenic content, total inorganic arsenic, and AS3 proportion.

[0087] The experimental data showed that arsenic pollution in millet is mainly inorganic arsenic (AS3), and there are significant batch-to-batch differences. The total arsenic and inorganic arsenic content in sample 12 is particularly high, and special attention should be paid to its source and possible health risks. In addition, the DMA content is relatively low and stable, while the ASV content fluctuates greatly between different samples, especially showing a significant increase in highly polluted samples. These findings are of great significance for the formulation of prevention and control measures for arsenic pollution in millet.

Claims

1. A method for detecting various arsenic forms in grains by pressurized microwave extraction, characterized in that: The following steps are involved: (1) Sample pretreatment: weigh 0.5 g sample, add 5 mL of 0.2% hydrochloric acid, extract for 20 min, extract at 80 °C, and auxiliary extraction pressure at 25 bar. After the extraction tube cools to room temperature, centrifuge at 8000 r for 5 min, take the supernatant, and filter using a 0.22 μm filter membrane to obtain the extract; (2) Preparation of standard samples: prepare standard solutions of hydrochloric acid and arsenic; (3) Detection: Liquid chromatography-inductively coupled plasma-mass spectrometry was used to detect the extract. The dual-column removal technology was used, with an AG7 chromatographic column connected before the six-way valve and another AG7 chromatographic column connected between the metering pump and the quantitative loop. The size of the AG7 chromatographic column was 2*50 mm. The conditions of liquid chromatography are: flow rate 0.4 ml / min, injection volume 5 μL, mobile phase elution conditions: 。 2. The method for detecting various arsenic forms in grain by pressurized microwave extraction-assisted detection according to claim 1, characterized in that: The parameters of the mass spectrometer are as follows: arsenic detection uses dual mass number detection, the first-stage mass number is 75, the second-stage mass number is 91, and the oxygen flow rate is 0.45 mL / min.

3. The method for detecting various arsenic forms in grain by pressurized microwave extraction-assisted detection according to claim 1, characterized in that: The forms of arsenic are: arsenic acid, arsenite, betaine, dimethyl arsenic, arsenocholine, monomethyl arsenic, p-ureidophenylarsonic acid, nitrophenylarsonic acid, p-nitrophenylarsonic acid, and roxarsone.

Images