Liquid chromatography-inductively coupled plasma mass spectrometry detection method for arsenic form in urine

Abstract

The invention relates to a liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) detection method for arsenic forms in urine, which comprises the following steps: (S1) respectively preparing single standard solutions of all arsenic forms, and diluting the single standard solutions to prepare a mixed standard solution; diluting the mixed standard solution step by step by using a 1-2mmol / L nitric acid solution; (S2) establishing a standard curve according to the mixed standard solution in the step (S1) by adopting liquid chromatography-inductively coupled plasma mass spectrometry; the mobile phase is 35-40 mmol / L ammonium carbonate and 1-2% methanol (v / v); a liquid chromatographic column is an alkyl quaternary ammonium salt anion chromatographic column; and (S3) diluting the urine sample with 1-2mmol / L nitric acid until the concentration of each arsenic form substance in the urine sample does not exceed the upper limit of the standard curve, testing each arsenic form substance in the urine sample under the same liquid chromatography-inductively coupled plasma mass spectrometry condition in the step (S2), and obtaining the content of each arsenic form substance in the urine according to the standard curve.

Description

Technical Field

[0001] The invention belongs to the technical field of arsenic form detection, and specifically relates to a liquid chromatography-inductively coupled plasma mass spectrometry detection method for arsenic forms in urine. Background Art

[0002] Arsenic, commonly known as arsenic, has the element symbol As. It is located in the 4th period and the VA group in the periodic table of chemical elements, with an atomic number of 33. The single substance exists in the form of three allotropes: gray arsenic, black arsenic and yellow arsenic. Arsenic is a natural environmental pollutant. Humans are usually exposed to arsenic-containing water, air, soil and food. The toxicity of arsenic is closely related to its form. According to the median lethal dose (LD50) published by the International Agency for Research on Cancer, the toxicity of arsenic is closely related to its form. 50 ), the toxicity of arsenic forms is as follows: arsenite (As(III))> arsenate (As(V))> monomethyl arsenic (MMA)> dimethyl arsenic (DMA), among which inorganic arsenic (As(III) and As(V)) are much more toxic than organic arsenic, while arsenocholine (AsC) and arsenobetaine (AsB) are considered to be basically non-toxic. Long-term exposure to inorganic arsenic can cause a variety of diseases, such as hypertension, skin cancer, lung cancer, etc., causing various hazards to human health. The "National Food Safety Standard Limits of Contaminants in Food" (GB 2762-2022) stipulates the limits of total arsenic and inorganic arsenic in food, so arsenic form analysis is crucial for food safety and human health. The effects on health of humans after exposure to arsenic forms of different toxicity vary greatly. In order to better evaluate the impact of arsenic on human health through food and environmental exposure pathways, it is necessary to conduct analytical studies on arsenic forms and arsenic metabolites in human urine. Therefore, it is very important to establish a method for analyzing multiple arsenic forms in urine.

[0003] At present, the main methods for detecting arsenic forms in urine are high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS), liquid chromatography-atomic fluorescence spectrometry (LC-AFS), hydride generation-atomic absorption spectrometry (LC-HG-AAS), ion exchange chromatography-hydride generation-atomic fluorescence spectrometry (LC-HG-ICP-AFS), etc. my country's health standard "Determination of Arsenic Forms in Urine" (WS / T 635-2018) uses LC-AFS to analyze arsenic forms in urine, but this standard can only determine four arsenic forms in urine (MMA, DMA, As(III), As(V)), and the sensitivity is relatively low. HPLC-ICP-MS has high sensitivity, a wide range of analysis types, and good separation, and is widely used in arsenic form analysis. At present, domestic and foreign researchers have adopted different analytical methods for the determination of arsenic forms in urine using HPLC-ICP-MS. The relevant literature is shown in Table 1. However, the relevant studies have problems such as long analysis time, complex experimental conditions, easy transformation of arsenic forms during pretreatment, and incomplete coverage of arsenic forms, which leads to low detection limits or large standard deviations (RSD). In order to better study the metabolic transformation of arsenic in urine after exposure, it is urgent to establish analytical methods for multiple arsenic forms in urine to evaluate the metabolic level after arsenic exposure. Table 1 is a brief introduction to different HPLC-ICP-MS methods for testing arsenic forms recorded in existing technical literature.

[0004] Table 1 Different analytical methods for the determination of arsenic forms in urine by HPLC-ICP-MS

[0005]

[0006]

[0007] The above prior art reports the methods of HPLC-ICP-MS determination of arsenic forms in urine under different conditions, generally using dihydrogen phosphate, monohydrogen phosphate, bicarbonate, and carbonate as mobile phases. The detection limit and sensitivity are different. However, the separation time is generally long, and at least about 10 minutes of detection time is required. In recent years, studies have also used acetate as a mobile phase, but its elution ability is poor and the analysis time is long.

[0008] The document "Rapid determination of six arsenic forms in urine by fully automated chromatography-tandem inductively coupled plasma mass spectrometry (LC-ICP-MS)", Jiang Jian et al., "Chinese Journal of Inorganic Analytical Chemistry", uses a fully automated chromatography-tandem inductively coupled plasma mass spectrometry to rapidly test six arsenic forms in urine, with mobile phase A being 0.5 mmol / L ammonium carbonate and mobile phase B being 80 mmol / L ammonium carbonate. According to the report, the detection time can be shortened to 2 minutes, and the detection limit is as low as 0.04-0.05 μg / L. However, based on the experimental conditions recorded, the experimental results could not be repeated.

[0009] In the article "Analysis of 8 Arsenic Forms in Human Urine by High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry" published by Wang Yiming and others in our research group, Shimadzu Shim-pack reverse phase chromatographic column was used. The mobile phase preparation of this method is relatively complex, and the chromatographic column has poor versatility. Compared with the method in the patent application, the sensitivity is equivalent, and the detection limit of the patent method is 0.15-0.20μg / L. The mobile phase preparation of the method of the present invention is relatively simple and convenient, and the chromatographic column used is Dion AS7 chromatographic column, which has good versatility and strong acid and alkali resistance.

[0010] In another article published by Liu Yang et al. in this research group, "Analysis of Six Arsenic Forms in Urine by High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry", the chromatographic column used in the article is the Dionex AS7 chromatographic column, and the elution method is gradient elution. This elution method will cause a gradient peak to appear at pentavalent arsenic, which will cause an error in the determination of pentavalent arsenic. In the method of the present invention, the gradient mobile phase elution method is changed to an elution method with different flow rates of the same mobile phase. Although the time is partially extended, the appearance of the gradient peak can be avoided, so that pentavalent arsenic can be accurately determined.

[0011] In addition, a problem that the existing technology cannot solve is that the signal intensity of arsenic choline (AsC) in urine is unstable under test conditions, and the stability of the test results is poor. At the same concentration, the response value of the AsC test result decreases over time, which leads to poor analytical stability of AsC and cannot provide accurate and reliable test results. Summary of the invention

[0012] In order to overcome the defects of the prior art of using liquid chromatography-inductively coupled plasma mass spectrometry to determine the arsenic forms in urine, such as poor detection effect, long detection time, and unstable AsC detection results, the present invention proposes a liquid chromatography-inductively coupled plasma mass spectrometry detection method for arsenic forms in urine, which determines six arsenic forms such as As(III), As(V), MMA, DMA, AsC, and AsB in urine. The six arsenic forms have a good linear relationship in the range of 0 to 150 μg / L, and the correlation coefficient (r) is greater than 0.999. The detection limit is 0.15 to 0.20 μg / L, and the relative standard deviation (RSD) of arsenic forms at different concentration levels is less than 3.0%. The spiked recovery rates at three different concentration levels are 96% to 107%. In addition, the present application can stabilize the AsC detection results by adding a small amount of nitric acid. Specifically, the present invention provides the following technical solutions to achieve the above-mentioned purpose:

[0013] A method for detecting arsenic forms in urine by liquid chromatography-inductively coupled plasma mass spectrometry comprises the following steps:

[0014] (S1) preparing single standard solutions of As(III), As(V), MMA, DMA, AsC, and AsB respectively, and then diluting the single standard solutions to prepare a mixed standard solution containing As(III), As(V), MMA, DMA, AsC, and AsB; and the mixed standard solution is diluted stepwise with a 1-2 mmol / L nitric acid solution;

[0015] (S2) using liquid chromatography-inductively coupled plasma mass spectrometry to establish a standard curve based on the mixed standard solution of step (S1); the mobile phase is 35-40 mmol / L ammonium carbonate + 1-2% methanol (v / v); the liquid chromatography column is an alkyl quaternary ammonium salt anion chromatography column;

[0016] (S3) diluting the urine sample with 1-2 mmol / L nitric acid until the concentration of each arsenic form in the urine sample does not exceed the upper limit of the standard curve; testing each arsenic form in the urine sample under the same liquid chromatography-inductively coupled plasma mass spectrometry conditions as in step (S2); and obtaining the content of each arsenic form in the urine according to the standard curve.

[0017] The inventor unexpectedly found that adding an appropriate amount of nitric acid to the sample can significantly improve the unstable phenomenon of AsC detection. The possible reason is that the inventor believes that the quaternary ammonium salt anion chromatographic column is adopted. The quaternary ammonium group in the quaternary ammonium salt is positively charged and is an ion exchange group with strong alkalinity. Dissociation occurs in the aqueous solution to form positively charged quaternary ammonium ions and corresponding anions. When the solution containing arsenic anions passes through the exchange column, the arsenic anions in the solution will exchange with the exchangeable anions on the quaternary ammonium salt exchange group. Based on the affinity difference between different anions and the quaternary ammonium salt exchange group, the movement speed of each arsenic anion in the exchange column is different during adsorption and elution, thereby achieving separation. Adding a small amount of nitric acid to the arsenic form standard solution may be conducive to improving the adsorption and desorption of AsC in the anion chromatographic column, thereby ensuring the stability of AsC.

[0018] Furthermore, the alkyl quaternary ammonium salt anion chromatographic column is selected from Dionex AS7 chromatographic column, Dionex AS14 chromatographic column, and Dionex AS19 chromatographic column.

[0019] Wherein, As(III) represents arsenite, As(V) represents arsenate, MMA represents monomethylarsenic, DMA represents dimethylarsenic, AsC represents arsenocholine, and AsB represents arsenobetaine. 3 3- ), arsenate (AsO 4 3- ), monomethylarsenic (CH 3 AsO 3 2- ), dimethylarsenic (C2 H 7 AsO 2 ), Arsenic choline (C 5 H 14 AsBrO) and arsenobetaine (C 5 H 11 AsO 2 ) are all certified reference materials.

[0020] Furthermore, in step (S1), the concentration of each form of arsenic standard substance in the single standard solution is 10.0-20.0 mg / L, and the concentration of each arsenic form substance As(III), As(V), MMA, DMA, AsC, and AsB in the mixed standard solution is the same; the mixed standard solution is diluted stepwise with 1-2 mmol nitric acid solution to 1.0 μg / L, 5.0 μg / L, 10.0 μg / L, 30.0 μg / L, 50.0 μg / L, 100.0 μg / L, and 200.0 μg / L.

[0021] Furthermore, in step (S1) and step (S3), the concentration of nitric acid used for dilution is 1.2-1.5 mmol / L.

[0022] Furthermore, in step (S2), the conditions of liquid chromatography are that the chromatographic column is a Dionex AS7 chromatographic column with a column length of 200-250 mm, an inner diameter of 4-5 mm, an injection volume of 20-30 μL, such as 25 μL; the gradient elution program is 0-2.99 min, and the flow rate is 0.7-0.8 L / min, 3 min-10 min, 1.2-1.3 L / min.

[0023] Further, in step (S2), the detection conditions of the inductively coupled plasma mass spectrometry are: RF power: 1200W~1550W, sampling depth of 6.0-8.0mm, carrier gas flow rate of 0.50-0.65L / min, compensation gas flow rate of 0.4-0.45L / min, collision gas flow rate of 4.5-5.0L / min, pump speed: 0.3-0.5r / s, integration time: 0.3-0.5s, detection mass-to-charge ratio (m / z): 75 and 35; preferably, the carrier gas and compensation gas are argon, and the collision gas is helium.

[0024] Further, in step (S3), the content (in terms of arsenic) of each arsenic form (AsB, DMA, As(III), AsC, MMA, As(V)) in the urine sample is calculated according to the following formula:

[0025] X i =(C i -C 0 )·K

[0026] Where:

[0027] X i - the content of arsenic forms in urine (measured as arsenic), in micrograms per liter (μg / L);

[0028] C i - the concentration of the arsenic form in the sample solution (measured in arsenic), in micrograms per liter (μg / L);

[0029] C 0 ——The concentration of arsenic species in the blank solution (measured in arsenic), in micrograms per liter (μg / L).

[0030] K——urine sample dilution multiple, the urine sample dilution multiple is 3-20 times, preferably 5-10 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the chromatogram of 20mmol / L ammonium carbonate + 1% methanol (mobile phase A), 100mmol / L ammonium carbonate + 1% methanol (mobile phase B), background blank and six arsenic forms mixed standard solution.

[0032] Figure 2 It is the chromatogram of 20mmol / L ammonium carbonate + 1% methanol (mobile phase A), 80mmol / L ammonium carbonate + 1% methanol (mobile phase B), background blank and six arsenic forms mixed standard solution.

[0033] Figure 3 It is the chromatogram of 20mmol / L ammonium carbonate + 1% methanol (mobile phase A), 60mmol / L ammonium carbonate + 1% methanol (mobile phase B), background blank and six arsenic forms mixed standard solution.

[0034] Figure 4 It is the chromatogram of 30mmol / L ammonium carbonate + 1% methanol (mobile phase A), 60mmol / L ammonium carbonate + 1% methanol (mobile phase B), background blank and six arsenic forms mixed standard solution.

[0035] Figure 5 It is the chromatogram of 30mmol / L ammonium carbonate + 1% methanol (mobile phase A), 45mmol / L ammonium carbonate + 1% methanol (B), background blank and six arsenic forms mixed standard solution.

[0036] Figure 6 It is the chromatogram of 35mmol / L ammonium carbonate + 1% methanol (mobile phase A), 45mmol / L ammonium carbonate + 1% methanol (mobile phase B), background blank and six arsenic forms mixed standard solution.

[0037] Figure 7 It is a chromatogram of a mixed standard solution of six arsenic forms in 45mmol / L ammonium carbonate + 1% methanol (mobile phase).

[0038] Figure 8 It is a chromatogram of a mixed standard solution of six arsenic forms in 35mmol / L ammonium carbonate + 1% methanol (mobile phase).

[0039] Fig. 9 It is a chromatogram of a mixed standard solution of six arsenic forms in 30mmol / L ammonium carbonate + 1% methanol (mobile phase).

[0040] Fig.10 It is a chromatogram of pentavalent arsenic under mobile phase conditions with different pH conditions (pH = 9.2 → 9.5 → 9.6 → 9.7 → 9.75 → 9.85 → 10 gradually increasing).

[0041] Fig.11 It is the chromatogram of the urine sample of Example 4.

[0042] Fig.12 This is the liquid chromatogram of 50μg / L mixed standard solution of six arsenic forms over preparation time.

[0043] Fig.13 It is a chromatogram of the dilution of the mixed standard solution of arsenic forms by adding different concentrations of nitric acid.

[0044] Fig.14 This is the chromatogram of the preparation time after adding 1.5mmol / L nitric acid to a 50μg / L mixed standard solution of six arsenic forms. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further explained and illustrated by specific embodiments below.

[0046] Arsenite (AsO 3 3- ), arsenate (AsO 4 3- ), monomethylarsenic (CH 3 AsO 3 2- ), dimethylarsenic (C 2 H 7 AsO 2 ), Arsenic Choline (C 5 H 14 AsBrO) and arsenobetaine (C 5 H 11 AsO 2 ) are all certified reference materials.

[0047] Liquid chromatography-inductively coupled plasma mass spectrometry (Agilent 1260-7700x).

[0048] All glassware in the present invention needs to be soaked in 20 vol% nitric acid solution for 24 hours, rinsed repeatedly with water, and finally rinsed with pure water.

[0049] Example 1

[0050] (S1) Accurately weigh the certified reference materials of arsenite, arsenate, monomethylarsenic, dimethylarsenic, arsenocholine, and arsenobetaine, and dilute them with water to prepare a single-label standard stock solution of arsenic form with an arsenic concentration of 10.0 mg / L (in terms of arsenic), and store them in a refrigerator at 2-8°C. The validity period is 6 months. Accurately pipette 1.00 mL of the six single-label standard stock solutions of arsenic forms with a concentration of 10.0 mg / L into a 10 mL volumetric flask, and dilute them to the mark with water. Accurately pipette a certain amount of the mixed standard solution of arsenic forms with a concentration of 1.0 mg / L, and dilute them step by step with 1.5 mmol / L nitric acid to 0.0 μg / L (1.5 mmol / L nitric acid solution as background blank), 1.0 μg / L, 5.0 μg / L, 10.0 μg / L, 30.0 μg / L, 50.0 μg / L, and 100.0 μg / L.

[0051] (S2) Liquid chromatography parameters:

[0052] Chromatographic column: AS7 chromatographic column; mobile phase is ammonium carbonate + 1% methanol; injection volume is 25 μL; gradient elution program is: 0-2.99 min, flow rate 0.8 mL / min; 3.00-10.00 min, flow rate 1.3 mL / min.

[0053] ICP-MS detection parameters:

[0054] RF power: 1200W~1550W; sampling depth: 8.0mm; carrier gas flow rate (argon): 0.65L / min; compensation gas flow rate (argon): 0.45L / min; collision gas flow rate (helium): 4.5mL / min; pump speed: 0.3r / s integration time: 0.5s; detection mass-to-charge ratio (m / z): 75 and 35.

[0055] According to the above-mentioned liquid chromatography-inductively coupled plasma mass spectrometry test parameters, the mixed standard solutions of arsenic forms of different concentrations obtained in step (S1) are tested to draw a standard curve.

[0056] (S3) diluting the urine sample with 1.5 mmol / L nitric acid until the concentration of each arsenic form in the urine sample does not exceed the upper limit of the standard curve of step (S2); testing each arsenic form in the urine sample under the same liquid chromatography-inductively coupled plasma mass spectrometry conditions as step (S2); and obtaining the content of each arsenic form in the urine according to the standard curve.

[0057] In order to investigate different mobile phases, the chromatograms of (1) 20 mmol / L ammonium carbonate + 1% methanol (mobile phase A), 100 mmol / L ammonium carbonate + 1% methanol (mobile phase B), background blank and six arsenic forms mixed standard solutions were shown in Figure 2. Figure 1 (2) Chromatograms of 20mmol / L ammonium carbonate + 1% methanol (mobile phase A), 80mmol / L ammonium carbonate + 1% methanol (mobile phase B), background blank and six arsenic forms mixed standard solution are shown in Figure 2 (3) 20mmol / L ammonium carbonate + 1% methanol (mobile phase A), 60mmol / L ammonium carbonate + 1% methanol (mobile phase B), background blank and six arsenic forms mixed standard solution chromatograms as shown Figure 3 (4) 30mmol / L ammonium carbonate + 1% methanol (mobile phase A), 60mmol / L ammonium carbonate + 1% methanol (mobile phase B), background blank and six arsenic forms mixed standard solution chromatograms as shown Figure 4 As shown; (5) 30mmol / L ammonium carbonate + 1% methanol (mobile phase A), 45mmol / L ammonium carbonate + 1% methanol (B), background blank and six arsenic forms mixed standard solution chromatograms as shown Figure 5 (6) 35mmol / L ammonium carbonate + 1% methanol (mobile phase A), 45mmol / L ammonium carbonate + 1% methanol (mobile phase B), background blank and six arsenic forms mixed standard solution chromatograms as shown Figure 6 As shown; (7) 45mmol / L ammonium carbonate + 1% methanol (mobile phase) six kinds of arsenic forms mixed standard solution chromatogram as shown Figure 7 As shown; (8) 35mmol / L ammonium carbonate + 1% methanol (mobile phase) six kinds of arsenic forms mixed standard solution chromatogram as shown Figure 8 As shown; (9) 30mmol / L ammonium carbonate (mobile phase) six kinds of arsenic form mixed standard solution chromatogram as shown Fig. 9 shown.

[0058] pass Figures 1 to 6 Compared with the results of the previous study, the separation, sensitivity and separation time of the six arsenic forms were good using the gradient concentration elution method. However, due to the gradient concentration mobile phase elution method, solvent peaks will appear, pentavalent arsenic will appear in the background blank, and the pentavalent arsenic peak shape is poor, which affects the accurate quantification of pentavalent arsenic. Therefore, it is considered to shorten the difference in gradient mobile phase concentration to reduce the background blank pentavalent arsenic. However, as the difference in gradient mobile phase decreases, the background blank pentavalent arsenic concentration decreases, but multiple platforms appear in the blank solution, which affects the arsenic form peak. Therefore, the subsequent experiments use the isocratic mobile phase gradient flow rate.

[0059] In the process of gradient flow rate elution, AsB, DMA, As(III), and AsC are close to each other, so low flow rate is used for elution. MMA and As(V) are far away and have a late elution time, so high flow rate is used for elution. When the isocratic concentration of 45mmol / L is used, the separation of AsB, DMA, As(III), and AsC is slightly poor; when the concentration of 30mmol / L is used, the sensitivity of pentavalent arsenic is poor and the elution time is long. Therefore, the isocratic concentration of 35mmol / L is finally selected, that is, the mobile phase is 35mmol / L ammonium carbonate + 1% methanol.

[0060] Example 3

[0061] Since the retention time of pentavalent arsenic in this chromatographic column is relatively long, resulting in low sensitivity, we considered adjusting the pH of the mobile phase to allow pentavalent arsenic to elute earlier, and investigated the mobile phases at different pH values. Fig.10 This is the chromatogram of pentavalent arsenic under different pH conditions (pH=9.2→9.5→9.6→9.7→9.75→9.85→10 gradually increases) under mobile phase conditions. It can be seen that with the increase of pH, the peak time of pentavalent arsenic gradually advances. When it reaches pH=10, it can be seen that there is no obvious improvement compared with pH=9.8. At the same time, it is found that the blank background of pH 10 has a relatively large inverted peak, and the higher pH is harmful to the chromatographic column. Therefore, a mobile phase with a pH of about 9.8 is selected for the experiment.

[0062] Based on the above liquid chromatography conditions, the mobile phase was finally selected as 35mmol / L ammonium carbonate + 1% methanol (pH = 9.8); the mixed standard solution of arsenic forms was diluted with 1.5mmol / L nitric acid step by step to 0.0μg / L (1.5mmol / L nitric acid solution as background blank), 1.0μg / L, 5.0μg / L, 10.0μg / L, 30.0μg / L, 50.0μg / L, 100.0μg / L. The established standard curve is shown in Table 2 below.

[0063] Table 2 Linear range and equation of six arsenic forms standard defects

[0064]

[0065] Example 4

[0066] A urine sample was randomly selected and diluted with 1.5mmol / L nitric acid. The chromatogram was as follows: Fig.11 As shown in the figure, six kinds of arsenic forms mixed standard solutions at four concentration levels of 6.0, 45.0, 90.0, and 150.0 μg / L were added respectively, and the spike recovery test was carried out. The results are shown in Table 3. It can be seen from the results in the table that the spike recovery rates of the four concentration levels are all in the range of 95.0% to 110%, and the RSD is within 3.0%.

[0067] Table 3 Spiked recoveries and relative standard deviations of six arsenic forms (n=6)

[0068]

[0069]

[0070] Example 5

[0071] The inventors found that during the experiment using the established method, when pure water was used to prepare the mixed standard of arsenic forms, the response value of AsC at the same concentration gradually decreased with the increase of preparation time. The results are shown in Table 4, which led to poor linearity. Fig.12 This is the chromatogram of the 50μg / L mixed standard solution of six arsenic forms over the preparation time. Four needles were taken continuously, each needle lasting 10 minutes. It can be clearly seen that the AsC signal value decreased significantly over time, and the intensity of the other five arsenic peaks changed little. This shows that the stability of AsC measurement is poor and the accuracy cannot be guaranteed.

[0072] Table 4 AsC signal intensity (50 μg / L)

[0073] time AsC signal strength (CPS) Reduction percentage 10min 88177 / 20min 71411 19.0％ 30min 57751 34.5％ 40min 48993 44.4％

[0074] In order to solve this problem, the inventors found that adding an appropriate amount of nitric acid during the preparation of the standard solution significantly improved the stability of AsC detection. Pure water, 0.5mmol / L nitric acid, 1mmol / L nitric acid, 1.5mmol / L nitric acid, 2mmol / L nitric acid, and 5mmol / L nitric acid were used to dilute the mixed standard solution of arsenic forms. The chromatogram is shown in Figure 2. Fig.13 As shown, it can be seen that with the increase of acidity, AsC is eluted more fully and the sensitivity gradually increases. However, after the acidity reaches 2mmol / , the sensitivity of AsC decreases. On this basis, the acidity is increased, and the sensitivity of AsC gradually increases, but the separation degree with As(III) is poor. Based on the above experiments, a nitric acid concentration of 1-2mmol / L, preferably 1.5mmol / L, is selected to prepare the mixed standard solution of arsenic forms. Fig.14 This is a liquid chromatogram of 10 needles (10min to 100min, 10min each needle) after adding 1.5mmol / L nitric acid to a 50μg / L mixed standard solution of six arsenic forms. It can be seen that after adding 1.5mmol / L nitric acid to the sample, the detection stability of AsC is significantly improved and basically does not change within 100min.

[0075] Example 6

[0076] According to the method of Example 4, a urine sample was selected and divided into 4 parts, wherein the experimental group 1 and the experimental group 2 were diluted with 1.5mmol / L nitric acid, and six arsenic forms were added to the mixed standard solutions (containing 1.5mmol / L nitric acid) at four concentration levels of 6.0μg / L, 45.0μg / L, 90.0μg / L, and 150.0μg / L, respectively; the experimental group 3 and the experimental group 4 were diluted with ultrapure water, and six arsenic forms were added to the mixed standard solutions (containing no nitric acid, i.e., the mixed standard solution was diluted with ultrapure water instead of 1.5mmol / L nitric acid) at four concentration levels of 6.0μg / L, 45.0μg / L, 90.0μg / L, and 150.0μg / L, respectively. Experimental groups 1 and 3 were prepared and tested immediately, and experimental groups 2 and 4 were tested after being stored for 60min at 25±2℃ and 65±5rH%. It was found that except for the large difference in the AsC results, the detection results of the other five arsenic forms were basically consistent, as shown in Table 5 below. The RSDs of experimental groups 1 and 2 with 1.5mmol / L nitric acid added were low, while the RSDs of experimental groups 3 and 4 without 1.5mmol / L nitric acid added were high. The addition of 1.5mmol / L nitric acid to the mixed standard solution of arsenic forms significantly improved the detection accuracy and sensitivity of AsC, and even after storage for 60 minutes, the detection accuracy and sensitivity were still good.

[0077] Table 5 AsC detection results (n = 6)

[0078]

Claims

1. A method for detecting arsenic forms in urine by liquid chromatography-inductively coupled plasma mass spectrometry, characterized in that: The following steps are involved: (S1) preparing single standard solutions of As(III), As(V), MMA, DMA, AsC, and AsB respectively, and then diluting the single standard solutions to prepare a mixed standard solution containing As(III), As(V), MMA, DMA, AsC, and AsB; and the mixed standard solution is diluted stepwise with a 1-2 mmol / L nitric acid solution; (S2) using liquid chromatography-inductively coupled plasma mass spectrometry to establish a standard curve based on the mixed standard solution of step (S1); the mobile phase is 35-40 mmol / L ammonium carbonate + 1-2% methanol, v / v; the liquid chromatography column is an alkyl quaternary ammonium salt anion chromatography column; (S3) diluting the urine sample with 1-2 mmol / L nitric acid until the concentration of each arsenic form in the urine sample does not exceed the upper limit of the standard curve; testing each arsenic form in the urine sample under the same liquid chromatography-inductively coupled plasma mass spectrometry conditions as in step (S2); and obtaining the content of each arsenic form in the urine according to the standard curve.

2. The detection method according to claim 1, characterized in that: The alkyl quaternary ammonium salt anion chromatographic column is selected from Dionex AS7 chromatographic column, Dionex AS14 chromatographic column and Dionex AS19 chromatographic column.

3. The detection method according to claim 1, characterized in that: In step (S1), the concentration of each arsenic form standard substance in the single standard solution is 10.0-20.0 mg / L, and the concentration of each arsenic form substance As(III), As(V), MMA, DMA, AsC, and AsB in the mixed standard solution is the same.

4. The detection method according to claim 1, characterized in that: The mixed standard solution was diluted stepwise with 1-2 mmol nitric acid solution to 1.0 μg / L, 5.0 μg / L, 10.0 μg / L, 30.0 μg / L, 50.0 μg / L, 100.0 μg / L, and 200.0 μg / L.

5. The detection method according to claim 1, characterized in that: In step (S1) and step (S3), the concentration of nitric acid used for dilution is 1.2-1.5 mmol / L.

6. The detection method according to claim 1, characterized in that: In step (S2), the conditions of liquid chromatography are: the chromatographic column is a Dionex AS7 chromatographic column with a column length of 200-250 mm, an inner diameter of 4-5 mm, and an injection volume of 20-30 μL; the gradient elution program is 0-2.99 min, and the flow rate is 0.7-0.8 L / min, 3 min-10 min, and 1.2-1.3 L / min.

7. The detection method according to claim 1, characterized in that: In step (S2), the detection conditions of inductively coupled plasma mass spectrometry are: radio frequency power: 1200W-1550W, sampling depth: 6.0-8.0mm, carrier gas flow rate: 0.50-0.65L / min, compensation gas flow rate: 0.4-0.45L / min, collision gas flow rate: 4.5-5.0L / min, pump speed: 0.3-0.5r / s, integration time: 0.3-0.5s, detection mass-to-charge ratio (m / z): 75 and 35.

8. The detection method according to claim 7, characterized in that: The carrier gas and compensation gas were argon, and the collision gas was helium.

9. The detection method according to claim 1, characterized in that: In step (S3), the content of each arsenic form (AsB, DMA, As(III), AsC, MMA, As(V)) in the urine sample is calculated in terms of arsenic according to the following formula: X i =(C i -C0)·K Where: X i ——The concentration of arsenic forms in urine samples, in μg / L; C i ——The concentration of arsenic species in the sample solution, in μg / L; C0——the concentration of arsenic species in the blank solution, in μg / L; K——dilution factor of urine sample.

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