Method for analyzing chemical forms of chromium, arsenic, cadmium, mercury and lead in sample based on HPLC-ICP-MS (High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry) combined technology

Abstract

The invention discloses a method for analyzing chemical forms of chromium, arsenic, cadmium, mercury and lead in a sample based on an HPLC-ICP-MS (High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry) combined technology. The method can realize simultaneous separation of 12 forms including Cr (III), As (III), As (V), MMA, DMA, Cd (II), Hg (II), MeHg, EtHg, Pb (II), TML and TEL at one time, the detection limit is low, the precision and accuracy are good, the used mobile phases are aqueous phases, and the green chemistry concept is met; the method disclosed by the invention can be used for simultaneously and rapidly detecting different chemical forms of chromium, arsenic, cadmium, mercury and lead in a culture fish sample or an environmental water sample.

Description

Technical Field

[0001] This invention relates to a method for detecting toxic metal (metalloid) contaminants in samples, specifically a method for analyzing the chemical speciation of chromium, arsenic, cadmium, mercury, and lead in farmed fish or water samples based on HPLC-ICP-MS. Background Technology

[0002] Chromium, arsenic, cadmium, mercury, and lead are several common toxic metallic (metalloid) pollutants that easily accumulate in organisms and amplify in the human body through the food chain, posing health risks. With advancements in research, researchers have gradually realized that the toxicity, mobility, and bioavailability of heavy metals are determined not only by the total amount of the metal element but also, to a greater extent, by its different chemical forms. Different forms produce different environmental effects; specific elemental forms of metals can impact the environment within certain concentration ranges.

[0003] Chromium (Cr) occupies a special place in organisms; depending on its form, it can be essential or toxic. Trace amounts of Cr(III) are essential nutrients for the human body, participating in the metabolism of carbohydrates, lipids, and proteins. However, Cr(III) must bind to suitable organic ligands; otherwise, free Cr(III) ions appear to lack biological activity. In contrast, Cr(VI) is highly toxic due to its high oxidation potential and ability to penetrate biological membranes. Cr(VI) (actually CrO4) 2- Ions can freely diffuse into cells through the cell membrane because their structure is similar to that of anions (such as SO42-). 2- Or PO4 3- Similar to Cr(III), Cr(VI) can be transported through suitable anion exchange channels, while Cr(III) cannot enter cells through the same ion channels. Therefore, Cr(VI) compounds are 1000 times more cytotoxic than Cr(III).

[0004] Arsenic (As) is chemically classified as a metalloid, possessing properties of both metallic and nonmetallic elements. It exists primarily in the environment in inorganic forms (e.g., As(III) and As(V)), with small amounts existing in organic forms. Common organic forms of arsenic include monomethylarsonic acid (MMA), dimethylarsonic acid (DMA), and arsenic betaine (AsB). Except for a few bacteria that can utilize arsenic compounds for energy, arsenic is toxic to most organisms. However, not all forms of arsenic are toxic; its toxicity is also related to its solubility, which is affected by pH and redox changes. Long-term exposure to arsenic increases the risk of diabetes, adverse pregnancy outcomes (APO), and even skin, lung, and bladder cancer.

[0005] Cadmium (Cd) is one of the most toxic metals in the environment, primarily produced through two pathways: natural release and anthropogenic factors. Cadmium mainly enters the body through the respiratory and digestive tracts and is absorbed by the lungs and intestines. Cd can form complexes with certain organic compounds such as metallothionein (MT), albumin, other plasma proteins, or peptides (such as glutathione), accumulating primarily in the liver and kidneys. Cd can damage various organ systems, including the respiratory, skeletal, reproductive, immune, and nervous systems, as well as the liver and kidneys. Once cadmium is absorbed by proximal tubular (PT) cells of the kidney, it induces MT, which is then chelated, inactivated, and stored in the cells.

[0006] Mercury (Hg) is a persistent toxic element that affects human and ecosystem health. It can be transported through air and water, undergoes various transformations, and is bioaccumulated and biomagnified in the food chain. Mercury exists in three main forms: metallic elemental form (Hg(0)), inorganic form (Hg(I) and Hg(II)), and organic form (MeHg, EtHg, etc.). Chronic exposure to Hg(0) can cause vomiting, abdominal pain, renal tubular necrosis, mood changes, and cognitive impairment, while acute exposure to Hg(0) can cause severe lung and neurological damage. Acute exposure to inorganic mercury or mercury salts can cause vomiting, hematochezia, hypovolemic shock, and severe abdominal pain. Methylmercury poisoning can have harmful effects on the central nervous system, leading to impaired motor coordination, visual and tactile sensory system dysfunction, and in severe cases, paralysis.

[0007] Lead (Pb) is a persistent toxic heavy metal widely distributed in nature, and its toxicity varies greatly depending on its form. Lead in the environment mainly originates from coal combustion, paint, cosmetics, etc. Long-term exposure to lead can lead to poisoning. However, lead is widely used as an anti-knock additive in gasoline due to its high flexibility, tensile strength, durability, and corrosion resistance. Therefore, trimethyllead chloride (TML), triethyllead chloride (TEL), and tetraethyllead are the main sources of organic lead pollution in the environment. Many researchers have shown that TML and TEL are much more toxic than inorganic lead (Pb(II)). Lead enters the human body through the food chain, damaging the immune and digestive systems. Furthermore, lead damages nerve cells in the nervous system, induces apoptosis, interferes with the storage and release of neurotransmitters, and leads to lipid peroxidation damage.

[0008] The toxicity, migration, and biological effects of chromium, arsenic, cadmium, mercury, and lead all depend on their chemical forms. Therefore, qualitative and quantitative analysis of the forms in which these substances exist is crucial. In recent years, HPLC-ICP-MS has been widely used in substance analysis due to its unique advantages, including simple coupling, ease of operation, wide linear range, and low detection limits.

[0009] This invention establishes a method for analyzing the chemical speciation of chromium, arsenic, cadmium, mercury, and lead in samples using HPLC-ICP-MS. When used for analyzing farmed fish samples, the precision reaches within 2.7%, and the sample spike recovery rate is above 81%. Since the concentrations of different species in water samples are often at low levels, this invention incorporates online solid-phase extraction (online-SPE) technology for water sample analysis, achieving a precision within 2.8% and a sample spike recovery rate above 83%. This invention provides technical support for the determination of chromium, arsenic, cadmium, mercury, and lead in farmed fish or water samples. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention proposes for the first time a method for analyzing the chemical speciation of chromium, arsenic, cadmium, mercury, and lead in samples using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS).

[0011] This invention enables the simultaneous analysis of different chemical forms of chromium, arsenic, cadmium, mercury, and lead in samples. Specifically, chromium forms include: inorganic Cr(III); arsenic forms include: inorganic As(III) and As(V), and organic MMA (monomethylarsonic acid) and DMA (dimethylarsonic acid); cadmium forms include: inorganic Cd(II); mercury forms include: inorganic Hg(II), and organic MeHg (methylmercury) and EtHg (ethylmercury); lead forms include: inorganic Pb(II), and organic TML (trimethyllead chloride) and TEL (triethyllead chloride).

[0012] The technical solution of the present invention is as follows:

[0013] A method for analyzing the chemical speciation of chromium, arsenic, cadmium, mercury, and lead in farmed fish or water samples using HPLC-ICP-MS, comprising:

[0014] (1) Sample pretreatment

[0015] When the sample is a farmed fish sample, the pretreatment method is as follows:

[0016] The farmed fish sample was mixed with the extract, vortexed and sonicated, centrifuged, and the supernatant was adjusted to pH 7 and filtered through a 0.22 μm filter membrane to obtain the sample test solution, which was then analyzed by HPLC-ICP-MS.

[0017] The extract was an aqueous solution containing 0.1 M HNO3 and 5 mM cysteine ​​(Cys);

[0018] The optimal mass-to-volume ratio of farmed fish sample to extract is 1:2.5, g / mL;

[0019] The preferred method is to vortex for 3 min, sonicate at 40℃ for 30 min, and centrifuge at 4000 rpm for 15 min;

[0020] When the sample is a water sample, the pretreatment method is as follows:

[0021] The water sample was filtered through a 0.45 μm filter membrane and subjected to online solid-phase extraction, including column pre-equilibration, sample enrichment, and sample elution. The resulting eluent was analyzed by HPLC-ICP-MS.

[0022] The conditions for online solid-phase extraction are: enrichment column: XB-C 18 (5μm, 10mm × 4.6mm id); Pre-equilibration agent: aqueous solution containing 1.0mM tetrabutylammonium hydroxide (TBAH) and 0.01mM dodecyltrimethylammonium bromide (CTAB); Eluent: 0.5% HNO3; Sample pH: 5.0–8.0; Enrichment flow rate: 10mL / min –1 ;

[0023] (2) Establishing a standard curve

[0024] Weigh out standard samples of different chemical forms of chromium, arsenic, cadmium, mercury, and lead: Cr(III), As(III), As(V), MMA, DMA, Cd(II), Hg(II), MeHg, EtHg, Pb(II), TML, and TEL, prepare mixed standard solutions, and dilute them stepwise to obtain a series of standard working solutions. Analyze these solutions using HPLC-ICP-MS under the following conditions:

[0025] HPLC separation: AQ-C 18 Pillar: Yuexu AQ-C 18 (5μm, 200mm × 4.6mm id); Mobile phase: aqueous solution containing 5mM tetrabutylammonium hydroxide (TBAH) and 5mM cysteine ​​(Cys), pH = 4.7; Flow rate: 1.5mL min –1 ;

[0026] ICP-MS detection: Plasma RF power 1200W, cooling gas flow rate 13.02L / min –1 Auxiliary gas flow rate: 0.75 L / min –1 Atomizing gas flow rate: 0.85 L / min –1 All gases are high-purity argon;

[0027] A standard curve was plotted with the concentrations of each chemical form of chromium, arsenic, cadmium, mercury, and lead on the x-axis and the peak areas in the HPLC-ICP-MS chromatograms on the y-axis.

[0028] (3) Sample Analysis

[0029] The HPLC-ICP-MS analysis conditions for the pretreated farmed fish or water samples in step (1) are the same as in step (2). The peak areas in the obtained HPLC-ICP-MS chromatogram are substituted into the standard curve established in step (2) to obtain the contents of different chemical forms of chromium, arsenic, cadmium, mercury and lead in the farmed fish or water samples.

[0030] The main advantages of the technical solution of this invention are as follows:

[0031] (1) This invention proposes a method for simultaneously determining different chemical forms of chromium, arsenic, cadmium, mercury, and lead in farmed fish or water samples using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). This method can simultaneously separate 12 forms, including Cr(III), As(III), As(V), MMA, DMA, Cd(II), Hg(II), MeHg, EtHg, Pb(II), TML, and TEL. This invention enables the simultaneous and rapid detection of different chemical forms of chromium, arsenic, cadmium, mercury, and lead in farmed fish or environmental water samples.

[0032] (2) Compared with the HPLC-ICP-MS method for the analysis of metal speciation of chromium, arsenic, cadmium, mercury and lead (Table 1), the present invention can simultaneously analyze 12 speciations of chromium, arsenic, cadmium, mercury and lead in one run, and the mobile phase used is a green and environmentally friendly aqueous mobile phase that does not contain any organic solvents, thus meeting the concept of green and environmental protection.

[0033] (3) When used for trace substance analysis in environmental water samples, compared with other published enrichment methods (Table 2), the online solid-phase extraction technology introduced in this invention has the advantages of simple operation, high enrichment factor, low detection limit, and reusable adsorbent. Furthermore, this method can complete the simultaneous enrichment analysis of multiple elemental speciations in a relatively short time, with the enrichment factors for As(V) and MMA being significantly higher than those of other methods.

[0034] Table 1. Comparison of analytical performance of this invention with other methods (cultured fish samples)

[0035]

[0036]

[0037] Table 2. Comparison of analytical performance of this invention with other methods (water samples)

[0038]

[0039] Attached Figure Description

[0040] the following Figures 1 to 7 Attached figures for analysis of farmed fish samples. Figures 8-19 The attached figures are related to the water sample analysis.

[0041] Figure 1 Mobile phase screening diagram; (a) 4mM TBAH pH 5.0, (b) 4mM TBAH + 5mM Cys pH 5.0.

[0042] Figure 2 : Optimization diagram of mobile phase pH.

[0043] Figure 3 Optimization diagram of TBAH concentration in the mobile phase.

[0044] Figure 4 Optimization diagram of Cys concentration in the mobile phase.

[0045] Figure 5 Chromatograms showing the separation of chromium, arsenic, cadmium, mercury, and lead.

[0046] Figure 6 Chromatograms of GBW10068 and GBW10029 for speciation analysis.

[0047] Figure 7 : Speciation analysis of chromium, arsenic, cadmium, mercury and lead in farmed fish samples.

[0048] Figure 8 Flowchart of online solid-phase extraction experiment for water samples.

[0049] Figure 9 Screening diagram of adsorbents and pre-equilibration agents.

[0050] Figure 10 Screening chart of TBAH concentration in pre-equilibration agents.

[0051] Figure 11 Screening chart of CTAB concentration in pre-equilibration agents.

[0052] Figure 12 Pre-equilibration agent volume screening diagram.

[0053] Figure 13 : Eluent screening chart.

[0054] Figure 14 Sample pH screening diagram.

[0055] Figure 15 Sample volume screening chart.

[0056] Figure 16 Enrichment velocity screening diagram.

[0057] Figure 17 10ng L-1 Enrichment of cadmium, arsenic, mercury and lead was determined by standard chromatograms.

[0058] Figure 18 Typical chromatograms of four natural water CRMs after dilution.

[0059] Figure 19 : Speciation analysis of cadmium, arsenic, mercury and lead in aquaculture pond water. Detailed Implementation

[0060] The present invention is further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0061] Examples 1-5 below are case studies related to the analysis of farmed fish samples, and Examples 6-9 are case studies related to the analysis of water samples.

[0062] Example 1: Process of extracting farmed fish samples

[0063] Weigh 4g of sample into a 50mL centrifuge tube, add 10mL of extraction buffer (containing 0.1M HNO3 and 5mM Cys), vortex for 3min, then sonicate at 40℃ for 30min in an ultrasonic cleaner, followed by centrifugation at 4000rpm for 15min. Collect the supernatant in a 50mL centrifuge tube. Repeat the above steps, combine the supernatants from both extractions, adjust the pH of the supernatant to neutral with 10M KOH, filter through a 0.22μm filter membrane, and then proceed with analysis.

[0064] Example 2: Optimization of liquid chromatography separation conditions

[0065] (1) Selection of mobile phase

[0066] TBAH can electrostatically attract arsenic anions, enabling the separation of different arsenic forms; Cys can complex with mercury and lead to achieve separation. Therefore, TBAH and Cys were added to the mobile phase. The results are as follows: Figure 1 As a preferred option, TBAH and Cys were added to the mobile phase simultaneously for further optimization in subsequent experiments.

[0067] (2) Selection of mobile phase, its concentration, and pH value

[0068] Appropriate selection of the mobile phase plays an important auxiliary role in improving separation efficiency. This application investigated mobile phases with different pH values. Different forms of chromium, arsenic, cadmium, mercury, and lead species have different ionization constants, and their retention times on the chromatographic column mainly vary with the concentration and pH of the mobile phase. Under different pH conditions, the degree of ionization of each species varies, and their interaction with the chromatographic column changes, resulting in different retention times. The separation of various forms of chromatin using TBAH and Cys at a concentration of 5.0 mM and a pH range of 4.0-5.0 was investigated. Figure 2 As shown, the best separation effect can be obtained for all forms at a pH of 4.7.

[0069] This application investigated the concentrations of TBAH and Cys in the mobile phase, examining the separation of different speciations when TBAH concentrations ranged from 1.0 to 7.0 mM and Cys concentrations ranged from 0.1 to 7.0 mM. The results are as follows: Figure 3 and Figure 4 Preferably, the TBAH concentration is 5 mM and the Cys concentration is 5 mM. The chromatogram under optimal separation conditions is shown below. Figure 5 .

[0070] Example 3: Optimization of Extraction Conditions

[0071] Commonly used extraction reagents for As include HNO3, EDTA, and methanol; for Cr, EDTA is commonly used; for Hg, Cys, HNO3, and HCl are commonly used; for Pb, EDTA and HCl are commonly used; and for Cd, HCl is commonly used. Therefore, eight extraction solutions were prepared using Cys, HNO3, CH3OH, HCl, and EDTA (see Table 2). In addition, the mobile phase was used as the extraction solution to extract GBW10068 and GBW10029 standards. The extracts were filtered, diluted tenfold, and directly measured by ICP-MS. The extraction results are shown in Table 3. Considering the extraction effect, background level, and impact on chromatographic separation, 5 mM Cys + 0.1 M HNO3 was selected as the final extraction reagent. After extraction, the pH of the extracted solution was adjusted to neutral using 2 M HNO3 and 2 M KOH.

[0072] Table 3. Total extracts (mg / kg) of GBW10068 and GBW10029 extracted with different extracts. -1 )

[0073]

[0074]

[0075] Example 4: Confirmation of the determination of different forms of chromium, arsenic, cadmium, mercury and lead using HPLC-ICP-MS

[0076] (1) Determination of the linear range of the combined analytical method

[0077] Standard samples of chromium, arsenic, cadmium, mercury, and lead were prepared to a concentration of 10 mg / L. -1 The mixed standard solution was serially diluted to the desired concentration. The concentrations of the mixed standard solutions were: 0.5, 2, 10, 20, 50, 100, and 200 μg / L. -1The series of mixed standard solutions were analyzed for chromium, arsenic, cadmium, mercury, and lead speciation under the instrument operating conditions described above. The peak areas of chromium, arsenic, cadmium, mercury, and lead speciation were measured, and a standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The linear correlation coefficients were all better than 0.994.

[0078] (2) Determination of detection limit and quantitation limit using combined analytical methods

[0079] Using the blank reagent of the standard sample, and under the above instrument conditions, perform ten parallel determinations. Calculate the limit of detection based on three times the standard deviation of the blank signal, and calculate the limit of quantitation based on ten times the standard deviation.

[0080] (3) Precision determination of combined analytical methods

[0081] Precision was used to examine the reproducibility of the method. Standard samples of chromium, arsenic, cadmium, mercury, and lead were prepared to a concentration of 100.0 μg / L. -1 A mixed standard solution of varying concentrations was prepared. Chromium, arsenic, cadmium, mercury, and lead were analyzed using a mixed standard solution (10 μg / L) under the conditions described above. –1 Typical chromatograms of mixed solutions of chromium, arsenic, cadmium, mercury, and lead standards are shown below. Figure 5 The peak area was measured six times in parallel, and the RSD of each peak shape was used as the metric. The results showed that the RSD was less than 2.7% for all peak shapes. The results are shown in Table 4.

[0082] Table 4 Analytical performance of HPLC-ICP-MS

[0083]

[0084] (4) Accuracy determination of combined analytical methods

[0085] This invention uses standard reference materials and spike recovery rates to measure the accuracy of the method. The standard addition method for calculating spike recovery rate involves adding standard solutions of various speciations of chromium, arsenic, cadmium, mercury, and lead to the matrix sample at known concentrations. After extraction, separation, and detection, the concentrations are measured, and the percentage difference between the actual and theoretical concentrations is calculated as the spike recovery rate. Standard reference materials for elemental speciation analysis are relatively scarce; only a few food matrix standard reference materials have been certified for specific speciations such as methylmercury or inorganic arsenic. GBW10068 is one of the few typical standard reference materials certified for total As, Cd, Hg, and Pb in aquatic product matrices, while GBW10029 is a fish standard material certified for MeHg. Therefore, this paper selected GBW10068 and GBW10029 to verify the accuracy of the analytical method. GBW10068 and GBW10029 were extracted using the optimal extraction solvent (0.1 MH NO3 + 5 mM Cys), and the chromatograms of the extracts are shown below. Figure 6The results are shown in Table 5. In the GBW10068 standard reference material, the measured values ​​of total As (As(III) + DMA), Cd(II), total Hg (Hg(II) + MeHg), and Pb(II) are in good agreement with the certified values ​​for total elemental composition. In the GBW10029 standard reference material, the measured value of MeHg is also in good agreement with the certified value. As shown in Tables 5 and 6, the recoveries of all speciations in the standard reference materials and actual samples range from 81% to 117%, verifying the accuracy of the analytical method.

[0086] Table 5. Morphological analysis results of GBW10068 and GBW10029 (n=3)

[0087]

[0088] Note: a Total amount of elements; b Not detected.

[0089] Example 5: Speciation analysis of chromium, arsenic, cadmium, mercury and lead in cultured fish samples

[0090] The established method was applied to the speciation analysis of chromium, arsenic, cadmium, mercury, and lead in farmed fish samples. Chromatograms are shown below. Figure 7 The data is shown in Table 6.

[0091] Table 6. Speciation analysis of chromium, arsenic, cadmium, mercury and lead in farmed fish samples (μg kg) -1 (n=3)

[0092]

[0093]

[0094] The results showed that trace amounts of As(III) and MeHg were detected in all fish samples, with MeHg levels ranging from 0.4 to 4.4 μg / kg. -1 The As(III) content ranged from 39 to 525 μg / kg. -1 Between. Spiking recovery experiments were conducted on all farmed fish samples, with an As spike amount of 250 μg / kg. -1 The spiking amounts of Cd, Hg, Pb, and Cr were 100 μg kg. -1 The recoveries of all spiked samples ranged from 81% to 110%.

[0095] Example 6: Water Sample Experiment Procedure

[0096] Online SPE-HPLC-ICP-MS primarily achieves simultaneous enrichment and analysis of arsenic, cadmium, mercury, and lead speciation through switching between a six-way and a fourteen-way valve. The experiment mainly comprises three processes: online solid-phase extraction (SPE), high-performance liquid chromatography (HPLC), and ICP-MS detection. A detailed schematic diagram is shown below. Figure 8 .

[0097] The online SPE program mainly consists of three steps: column pre-equilibration, sample enrichment, and sample elution. The specific operation is as follows:

[0098] Column pre-equilibration: Both the high-pressure six-way valve (valve 1) and the high-pressure fourteen-way valve (valve 2) are in the "load" state. First, manually inject 8 mL of 1.0 mM MTABAH + 0.01 mM CTAB pre-equilibration agent into the metering loop of valve 2, switch the fourteen-way valve (valve 2) to the "inject" state, turn on the liquid phase pump (pump 2), and use ultrapure water at a rate of 10 mL / min. -1 The flow rate carries the pre-equilibrating agent into XB-C 18 In the enrichment column. After 1 minute, stop pump 2. Then switch valve 2 from "inject" to "load".

[0099] Sample enrichment: Manually inject 10 mL of standard solution into the metering loop of valve 2, switch valve 2 to "inject", and turn on pump 2 to carry the solution into XB-C. 18 Sample pre-enrichment was achieved in the enrichment column. After 1 minute, valve 2 was immediately switched to the "load" state, and pump 2 was simultaneously turned off. At this point, arsenic, cadmium, mercury, and lead species had been adsorbed onto the XB-C column. 18 Enriched in the column.

[0100] Sample elution: After the enrichment process is complete, switch valve 1 to "inject", turn on pump 1, and trigger the ICP-MS system to acquire data. After data acquisition is complete, turn off pumps 1 and 2, and switch both valves 1 and 2 to "load" state.

[0101] After elution with eluent, the sample is placed in a 20cm AQ-C immersion chamber. 18 The speciation was separated by chromatographic column, and then the speciations of arsenic, cadmium, mercury and lead were detected and analyzed by ICP-MS system.

[0102] Example 7: Optimization of Enrichment Conditions

[0103] (1) Selection of adsorbent and pre-equilibration agent

[0104] Based on the differences between adsorption and desorption mechanisms, C 18 Five adsorbents, namely SCX, SAX, Amphion II, and GO@SiO2, were screened. The results are as follows: Figure 9As shown, when XB-C18 is used as the adsorbent and 1.0 mM MTBAH + 0.01 mM CTAB is used as the pre-equilibration agent, the enrichment effect of each species of arsenic, cadmium, mercury and lead can be obtained.

[0105] (2) This application screened the concentration and volume of the pre-equilibration agent, as the concentration and volume of the pre-equilibration agent can affect the enrichment effect to a certain extent. In order to obtain the best enrichment effect, the concentration of TBAH in the range of 0.5-4 mM and the concentration of CTAB in the range of 0.001-0.01 mM were optimized. The results are shown in […]. Figure 10 and Figure 11 When the concentration of TBAH in the pre-equilibration agent is 1.0 mM and the concentration of CTAB is 0.01 mM, the enrichment effect is better.

[0106] The enrichment effects of each species were investigated at pre-equilibration agent volumes of 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, and 10.0 mL, respectively. The results are as follows: Figure 12 As shown, arsenic, cadmium, mercury and lead can be well enriched when the volume of the pre-equilibration agent is 8.0 mL.

[0107] (3) This application also screened the types of eluents. The type of eluent mainly affects the elution effect of each species. If the eluent is ineffective, the adsorbed species cannot be eluted from the enrichment column. Therefore, the enrichment effect of different concentrations of acid as eluents, such as... Figure 13 When using 10 μL of 0.5% HNO3 as the eluent, a better enrichment effect can be obtained.

[0108] (4) This application screened samples based on pH. Samples with pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 were screened, and the results are as follows: Figure 14 The enrichment effect is best when the pH range is 5.0-8.0.

[0109] (5) This application also screened the sample volume and enrichment flow rate. The enrichment effect was investigated when the sample volume was 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, and 10.0 mL, and the results are as follows: Figure 15 As shown, the enrichment factor increases with increasing sample volume; therefore, an injection volume of 10.0 mL was selected for subsequent experiments. The enrichment factor at 2.0 mL min was also investigated. -1 4.0 mL min -1 6.0 mL min -1 8.0 mL min -1 10.0 mL min -1 The effect of enrichment flow rate on enrichment effect. For example... Figure 16The enrichment flow rate had virtually no effect on the enrichment effect. To shorten the experimental time, a flow rate of 10.0 mL / min was ultimately chosen. -1 .

[0110] Example 8: Confirmation of the determination of different forms of arsenic, cadmium, mercury and lead using SPE-HPLC-ICP-MS

[0111] (1) Determination of the linear range of the combined analytical method

[0112] Arsenic, cadmium, mercury, and lead speciation standard samples were prepared to a concentration of 10 mg / L for As(III), As(V), DMA, MMA, Cd(II), Hg(II), MeHg, EtHg, Pb(II), TML, and TEL. -1 The standard solutions were serially diluted to the required concentrations. The series of mixed standard solutions for arsenic, cadmium, mercury, and lead were analyzed for arsenic, cadmium, mercury, and lead speciation under the instrument operating conditions described above (10 ng / L). –1 Typical chromatograms of mixed solutions of arsenic, cadmium, mercury, and lead standards are shown below. Figure 17 The peak areas of arsenic, cadmium, mercury, and lead were measured, and a standard curve was plotted with concentration on the x-axis and peak area on the y-axis. The linear correlation coefficients were all better than 0.995.

[0113] (2) Determination of detection limit and quantitation limit using combined analytical methods

[0114] Using the blank reagent of the standard sample, and under the above instrument conditions, perform ten measurements. Calculate the limit of detection based on three times the standard deviation of the blank signal, and calculate the limit of quantitation based on ten times the standard deviation.

[0115] (3) Precision determination of combined analytical methods

[0116] Precision was used to examine the reproducibility of the method. Standard samples of arsenic, cadmium, mercury, and lead were prepared to a concentration of 10.0 ng / L. -1 Mixed standard solutions of varying concentrations were prepared. Under the aforementioned working conditions, mixed standards of arsenic, cadmium, mercury, and lead were analyzed in six parallel determinations. The RSD was calculated based on the peak area of ​​each speciation. The results showed that the RSDs were all less than 2.8%. The determination results are shown in Table 7.

[0117] Table 7 Analytical performance of online SPE-HPLC-ICP-MS

[0118]

[0119]

[0120] (4) Accuracy determination of combined analytical methods

[0121] This invention uses standard reference materials and spike recovery rates to measure the accuracy of the method. The standard addition method for calculating spike recovery rate involves adding standard solutions of known concentrations of various speciations of chromium, arsenic, cadmium, mercury, and lead to the matrix sample. After enrichment, separation, and detection, the concentrations are measured, and the percentage relationship between the actual concentration difference and the theoretical value is calculated as the spike recovery rate. Standard reference materials for elemental speciation analysis are relatively scarce. No environmental water matrix standard reference materials have been certified for any speciation of As, Cd, Hg, and Pb. Therefore, this paper selected four standard reference materials certified for total elements in natural water: total Pb (GBW08601), total cadmium (GBW08602), total mercury (GBW08603), and total arsenic (GBW08695) for determination, and spike recovery was also performed. Because the standard material concentration was too high, GBW08601 was diluted 10% before measurement. 5 GBW08602 diluted 10 times 4 GBW08603 diluted 10 times 3 GBW08605 diluted 5×10 4 The typical chromatograms of the four standard substances after dilution are shown below. Figure 18 The speciation, content, and spiked recovery data are shown in Table 8. The data in Table 8 indicate that Pb(II) is the only present form in certified substance GBW08601, Hg(II) is the main present form in certified substance GBW08603, and As(III) is the main present form in certified substance GBW08605. The total values ​​of all elements measured are highly consistent with the certified values, and the spiked recoveries of all speciations are between 87% and 109%. As shown in Table 8, the spiked recoveries of the water samples are all above 83%.

[0122] Table 8. Speciation analysis of four types of natural water CRMs

[0123]

[0124] Note: a Total element certified value; b not detected.

[0125] Example 9: Analysis of mixed speciation of arsenic, cadmium, mercury and lead in water samples

[0126] The established method was applied to the speciation analysis of arsenic, cadmium, mercury, and lead in water samples. The chromatograms are shown below. Figure 19 The data is shown in Table 9.

[0127] Table 9. Speciation of arsenic, cadmium, mercury, and lead in water samples (ng / L) -1 (n=3)

[0128]

[0129]

[0130] The results showed that trace amounts of Pb(II), Cd(II), and Hg(II) were detected in all 24 water samples tested, with Pb(II) content ranging from 46.4 to 148.4 ng / L. -1 The Cd(II) content ranged from 9.2 to 24.5 ng / L. -1 The Hg(II) content ranged from 16.1 to 30.6 ng / L. -1 The results indicate that this method can be applied to the analysis and determination of low concentrations of toxic elements in environmental water. Spiking recovery experiments were performed on all water samples, with a spiked amount of 0.1 μg / L. -1 The spiked recoveries ranged from 83% to 116%.

Claims

1. A method for analyzing the chemical forms of chromium, arsenic, cadmium, mercury and lead in cultured fish samples or water samples based on HPLC-ICP-MS combined technology, characterized in that, The method comprises: (1) sample pretreatment When the sample is a farmed fish sample, the pretreatment method is as follows: Mix the farmed fish sample with the extraction solution, perform vortexing and ultrasonic treatment, centrifuge, take the supernatant, adjust the pH to 7, filter through a 0.22 mu m filter membrane, and obtain the sample to be tested liquid to enter the HPLC-ICP-MS instrument for analysis; The extraction solution is an aqueous solution containing 0.1M HNO3 and 5mM cysteine; When the sample is a water sample, the pretreatment method is as follows: Filter the water sample through a 0.45 mu m filter membrane, perform online solid phase extraction, including column pre-equilibration, sample enrichment, and sample elution, and obtain the sample eluent to enter the HPLC-ICP-MS instrument for analysis; (2) Establish a standard curve Weigh different chemical forms of standard samples of chromium, arsenic, cadmium, mercury, and lead: Cr(III), As(III), As(V), MMA, DMA, Cd(II), Hg(II), MeHg, EtHg, Pb(II), TML, and TEL, prepare a mixed standard solution, dilute it step by step to obtain a series of standard working solutions, and analyze them using the HPLC-ICP-MS instrument under the following conditions: HPLC separation: AQ-C 18 Column; mobile phase: aqueous solution containing 5 mM tetrabutylammonium hydroxide and 5 mM cysteine, pH = 4.7; flow rate: 1.5 mL min –1 ; ICP-MS detection: Plasma RF power 1200 W, cooling gas flow 13.02 L min –1 , auxiliary gas flow 0.75 L min –1 , atomizing gas flow 0.85 L min –1 , all gases are high-purity argon Use the concentrations of each chemical form of chromium, arsenic, cadmium, mercury, and lead as the abscissa, and the peak area in the HPLC-ICP-MS chromatogram as the ordinate, to draw a standard curve. (3) Sample analysis The HPLC-ICP-MS analysis conditions of the farmed fish sample or water sample after pretreatment in step (1) are the same as in step (2), the peak area in the obtained HPLC-ICP-MS chromatogram is substituted into the standard curve established in step (2), and the contents of different chemical forms of chromium, arsenic, cadmium, mercury, and lead in the farmed fish sample or water sample are obtained.

2. The method for analyzing the chemical forms of chromium, arsenic, cadmium, mercury and lead in the cultured fish sample or water sample based on the HPLC-ICP-MS combined technique according to claim 1, characterized in that, In the pretreatment process of the farmed fish sample in step (1), the mass-volume ratio of the farmed fish sample to the extraction solution is 1:2.5, g / mL.

3. The method for analyzing the chemical forms of chromium, arsenic, cadmium, mercury and lead in the cultured fish sample or water sample based on the HPLC-ICP-MS combined technique according to claim 1, characterized in that, In the pretreatment process of the farmed fish sample in step (1), vortex for 3 minutes, ultrasonic treatment at 40°C for 30 minutes, and centrifugation at 4000 rpm for 15 minutes.

4. The method for analyzing the chemical forms of chromium, arsenic, cadmium, mercury and lead in the cultured fish sample or water sample based on the HPLC-ICP-MS combined technique according to claim 1, characterized in that, Step (1) The conditions of on-line solid phase extraction during the pretreatment of water sample are as follows: enrichment column: XB-C 18 ; pre-equilibration agent: an aqueous solution containing 1.0 mM tetrabutylammonium hydroxide and 0.01 mM dodecyltrimethylammonium bromide; Eluent: mass fraction 0.5% HNO3; sample pH: 5.0-8.0; enrichment flow rate: 10 mL min –1 .

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