Arsenic derivative extraction and high performance liquid chromatography-inductively coupled plasma mass spectrometry combined technology determination method

Abstract

The invention discloses an arsenic derivative extraction and high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) determination method, and the arsenic derivative determination method comprises the following steps: 1) pretreating a to-be-determined sample containing an arsenic derivative, and preparing a to-be-determined sample solution; 2) carrying out high performance liquid chromatography-mass spectrometry analysis on the to-be-detected sample solution; wherein the mobile phase of the eluent of the high performance liquid chromatography elution is as follows: the mobile phase A is a mixed solution of 5% methanol and 5 mmol / L ammonium carbonate, and the mobile phase B is a mixed solution of 5% methanol and 100 mmol / L ammonium carbonate. According to the present invention, the detection method is optimized, the mobile phase concentration and the components are adjusted, the new detection method is established, the mobile phase and the gradient elution mode are adopted, the arsenic standard substances in six forms can be completely detected, the result is good, and the detection limit and the precision are good.

Description

Technical Field

[0001] The invention relates to a detection method, in particular to an arsenic derivative extraction and high performance liquid chromatography-inductively coupled plasma mass spectrometry combined technology determination method. Background Art

[0002] Arsenic (As) is a naturally occurring element that is widely present throughout the earth's crust and is released into natural water bodies through leaching and erosion of arsenic-containing minerals and soils. It can be divided into inorganic arsenic and organic arsenic. Inorganic arsenic is a highly toxic substance that is harmful to humans and the environment. The toxicity of arsenic is mainly manifested in two types: acute and chronic. Acute arsenic poisoning can cause symptoms such as nausea, vomiting, diarrhea, toxic shock, and in severe cases, death. Long-term exposure to low concentrations of arsenic compounds may cause chronic arsenic poisoning, the symptoms of which include skin lesions, respiratory diseases, nervous system damage, and cancer.

[0003] Methylated arsenic derivatives are organic compounds of arsenic, and common ones include methylarsenic acid (MMA) and dimethylarsenic acid (DMA). These organic arsenic compounds are slightly less toxic than inorganic arsenic compounds. They can enter the human body through food, drinking water, and air, and can be converted into each other through metabolism in the human body. Organic arsenic can be converted into other types of arsenic in animals, posing a potential threat to human health. The toxicity mechanism of methylated arsenic derivatives is not fully understood, but studies have shown that they may cause cell and tissue damage by affecting cell DNA repair, redox balance, apoptosis, and cell cycle. Methylated arsenic derivatives are also believed to be related to the occurrence and development of cancer. There are more than 100 arsenic compounds in the environment and biological systems, and the toxicity of arsenic varies greatly due to its chemical form and oxidation state. Studying the different forms and toxicological properties of arsenic compounds can better understand the distribution, transformation, toxicity of arsenic in the human body, as well as the metabolism and health effects of arsenic in biological systems.

[0004] Arsenic speciation analysis methods mainly use a combination of detection technologies, such as liquid chromatography-fluorescence, liquid chromatography-inductively coupled plasma mass spectrometry, etc. Due to its high sensitivity and high separation effect on different forms of arsenic, high performance liquid chromatography-inductively coupled plasma mass spectrometry is currently one of the effective methods for analyzing arsenic speciation.

[0005] Epidemiological studies have shown that exposure to low concentrations of arsenic is associated with the occurrence of various tumors such as skin cancer. Although arsenic is recognized as one of the human carcinogens, its carcinogenic mechanism is still controversial. Some scholars believe that arsenic can cause tumors alone, while others believe that arsenic only cooperates with other carcinogens to cause malignant transformation of normal cells. Summary of the invention

[0006] The object of the present invention is to provide a method for measuring the content of arsenic elements of various valences by high performance liquid chromatography-inductively coupled plasma mass spectrometry.

[0007] The present invention provides a method for determining arsenic derivatives, comprising the following steps: 1) Pre-treating the sample to be tested containing arsenic derivatives and preparing the sample solution to be tested; 2) The sample solution to be tested is analyzed by high performance liquid chromatography-mass spectrometry; The mobile phases of the elution solution for HPLC were as follows: mobile phase A was a mixture of 5% methanol and 5 mmol / L ammonium carbonate, and mobile phase B was a mixture of 5% methanol and 100 mmol / L ammonium carbonate.

[0008] Furthermore, the elution procedure of the high performance liquid chromatography is gradient elution.

[0009] Furthermore, the specific method of the gradient elution includes: 0-6 min, 100% mobile phase A; 6-10 min, 100% mobile phase A; 10-15 min, 100% mobile phase B; 15-20 min, 100% mobile phase B. The next cycle begins 20 min later.

[0010] Furthermore, the analysis method is high performance liquid chromatography-inductively coupled plasma mass spectrometry analysis.

[0011] Furthermore, the detection conditions of the mass spectrometer are: plasma power is 1550 W, the carrier gas is high-purity argon; the carrier gas flow rate is 1.0 L / min; the sampling depth is 5 mm; the cooling gas flow rate is 14 L / min; the auxiliary gas flow rate is 0.8 L / min; and the carrier gas flow rate is 0.9 L / min.

[0012] Furthermore, the sample to be tested is a cell containing an arsenic derivative; the pretreatment method of the sample to be tested is: adding a lysis solution to the sample to be tested to lyse the cells, shaking, centrifuging and filtering, and taking the filtrate as the sample solution to be tested.

[0013] Furthermore, the amount of the lysate added is 1×10 6 cell / 100 μL RIPA lysis buffer; the shaking time was 5 min; the centrifugal filtration method was to aspirate the cell solution into a centrifuge tube with a 3 kD retention membrane, and centrifuge and filter for 30 min at 4°C and 14000×g.

[0014] Furthermore, the sample to be tested is a marine sample; the pretreatment method of the sample to be tested is: adding the sample to be tested into a methanol-water solution, homogenizing at 8000 r / min for 2 min, and then ultrasonically extracting for 10 min; then transferring to a centrifuge tube, refrigerated centrifuging at 10000 r / min for 15 min; taking the supernatant and filtering it through a 0.22 μm filter membrane, and the obtained filtrate is the sample solution to be tested.

[0015] Furthermore, each gram of the marine sample is added into 10 mL of methanol-water solution, wherein the methanol-water solution is a solution with a volume ratio of 4:6.

[0016] The present invention optimizes the detection method, adjusts the mobile phase concentration and composition, and establishes a new detection method, using a mobile phase of 5% methanol + 5 mmol / L ammonium carbonate mixed solution (A), 5% methanol + 100mmol / L ammonium carbonate mixed solution (B), and a gradient elution method to completely detect six forms of arsenic standards, and the results are good, and the detection limit and precision are good. The present invention also explores that As (III) is converted into As (V) in melanoma cells within 24 hours, providing some evidence for the subsequent exploration of arsenic form detection methods and the detoxification mechanism of As (III) in melanoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The HPLC-ICP-MS standard chromatograms of six arsenic derivatives under isocratic elution conditions with different concentrations of mobile phase (10-100 mM (NH4)2CO3) (the elution order of the six forms is: AsB, DMA, As(Ⅲ), AsC, MMA and As(V)).

[0018] Figure 2 HPLC-ICP-MS chromatograms of six arsenic derivatives under gradient elution conditions with two gradient concentrations of mobile phase (5-100 mM vs. 10-100 mM (NH4)2CO3).

[0019] Figure 3 This figure shows the effect of adding methanol into the mobile phase on the HPLC-ICP-MS spectra of six standard arsenic derivatives.

[0020] Figure 4 This is the effect of mobile phase pH on the HPLC-ICP-MS spectra of six arsenic standard derivatives.

[0021] Figure 5 The HPLC-ICP-MS chromatogram of a 100 µg / L mixed standard solution of six arsenic forms.

[0022] Figure 6The HPLC-ICP-MS chromatogram of A375 cells.

[0023] Figure 7 This is the HPLC-ICP-MS chromatogram of seafood samples.

[0024] Figure 8 This is the HPLC working condition.

[0025] Fig. 9 The working conditions of ICP-MS.

[0026] Fig.10 Schematic diagram of mobile phase gradient elution program.

[0027] Fig.11 is the recovery rate of six arsenic forms under different extraction methods.

[0028] Fig.12 The following are the standard curves for six arsenic forms.

[0029] Fig.13 Detection limits of six arsenic species by HPLC-ICP-MS.

[0030] Fig.14 The precision of HPLC-ICP-MS for the detection of six arsenic species. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0032] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0033] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0034] 1. Instruments and reagents 1.1 Main instruments 1290 high performance liquid chromatograph (Agilent, USA); 8800 inductively coupled plasma mass spectrometer (Agilent, USA); Milli-Q water purifier (Merck, Germany); 5510 ultrasonic cleaner (Branson, USA); XR 205SM-DR precision electronic balance (Precisa, Switzerland); SevenMulti pH meter (Mettler Toledo, Switzerland); DKZ-2 constant temperature oscillator (Jinghong, Shanghai); Dionex IonPac AS 7 anion exchange column (ThermoFisher, USA); 5% CO2 humidified incubator (Thermo Scientific, USA); inverted microscope (Nikon, Japan); microwave digestion system (Aoton PAaron, Austria).

[0035] 1.2 Reagents and Materials Nitric acid (super pure, Merck, Germany); ammonium carbonate (analytical pure, Merck, Germany); methanol (LC-MS, Merck, Germany); arsenic trioxide ATO (Thermo Fisher, USA); 0.25% Trypsin-EDTA (Gibco, USA); Pierce TM RIPA Buffer (Thermo Fisher, USA); PBS buffer (Solarbio, China); fetal bovine serum (Gibco, USA); high glucose DMEM medium (Gibco, USA); penicillin-streptomycin (Thermo Fisher Company, USA); Arsenic standard solution: arsenite As(III) (GBW08666), arsenate As(V) (GBW08667), monomethylarsenic MMA (GBW08668), dimethylarsenic DMA (GBW08669), arsenocholine AsC (GBW08671), arsenobetaine AsB (GBW08670) were all purchased from the National Institute of Metrology, China; the laboratory water was ultrapure water (resistivity 18.2 MΩ.cm, MILLIPORE).

[0036] 1.3 HPLC-ICP-MS conditions Mass spectrometry conditions (optimized conditions): plasma power 1550 W, carrier gas high-purity argon; carrier gas flow rate 1.0 L / min; sampling depth 5 mm; cooling gas flow rate 14 L / min; auxiliary gas flow rate 0.8 L / min; carrier gas flow rate 0.9 L / min; data acquisition repeated 3 times, see Fig. 9 .

[0037] Chromatographic conditions (optimized conditions): Thermo Fisher AS7 anion exchange column (250 mm × 4.6 mm); mobile phase: 5 mmol / L ammonium carbonate (A), 100 mmol / L ammonium carbonate (B), gradient elution, elution program (see Figure 8 and Fig.10 ); flow rate was 1mL / min; injection volume was 20 μL.

[0038] 1. Optimization of arsenic derivative extraction and HPLC-ICP-MS detection method 2 Pre-treatment steps for samples 2.1 Cell sample processing steps 2.1.1 Cell culture The human malignant melanoma cell line A375 was purchased from Pronocell. A375 cells were routinely cultured in 7 mL of culture medium (89% high-glucose DMEM medium + 1% penicillin-streptomycin + 10% fetal bovine serum) and cultured in an incubator at 37°C and 5% CO2.

[0039] 2.1.2 Cell administration and collection When the cells grow to 80% confluence, add 7 μL of 10 μmol / mL arsenic trioxide solution prepared in serum-free DMEM and continue culturing in a 5% CO2 humidified incubator for 24 h.

[0040] Aspirate the culture medium, add PBS to wash the cells growing on the culture dish, remove PBS, and repeat the operation twice. Add 1 mL of trypsin to digest for 1 min, then add 2 mL of culture medium to terminate the digestion, gently blow the culture dish, collect the solution in the culture dish into a 15 mL centrifuge tube, centrifuge for 5 min (0.8 rpm), remove the supernatant, and collect the cells.

[0041] 2.1.3 Extraction of arsenic derivatives from cells Add 1×10 6 Cells were lysed with 100 μL RIPA lysis buffer and shaken for 5 min to fully lyse the cells. The cell suspension was pipetted into a centrifuge tube with a 3 kD cut-off membrane and centrifuged for 30 min (4°C, 14000×g). The filtrate was used for HPLC-ICP-MS detection.

[0042] 2.2 Seafood sample processing steps (see 5.4 Seafood sample optimization conditions for details) 2.2.1 Pretreatment of seafood samples The collected marine tissues were weighed and the wet weight was recorded. They were freeze-dried using a freeze dryer and the dry weight was recorded. They were then ground into powder using a pulverizer. The samples were then collected and placed in a drying dish for storage.

[0043] 2.2.2 Extraction of arsenic derivatives from seafood samples Specific steps: weigh about 1 g of sample and add it to a 15 mL centrifuge tube, add 10 mL of methanol-water (4:6, V / V) solution; homogenize at 8000 r / min for 1 min, then ultrasonically extract for 10 min; then transfer to a centrifuge tube, refrigerate centrifuge at 10000 r / min for 15 min; take the supernatant and filter it through a 0.22 μm filter membrane, and analyze the filtrate on the machine. The recovery rates of AsB, DMA, AsC, MMA and As(V) obtained can reach 95% or more, and the recovery rate of As(Ⅲ) is also higher than that of the previous experiment, reaching more than 75%.

[0044] 3. Preparation of standard solution Preparation of standard stock solution: Accurately weigh 0.3571 g, 0.2577 g, 0.1890 g, 0.1321 g, 0.3984 g and 0.5714 g of arseniccholine solution standard substance, 0.2577 g, 0.1890 g, 0.1321 g, 0.3984 g and 0.5714 g of dimethyl arsenic solution standard substance, arsenite solution standard substance, monomethyl arsenic solution standard substance and arsenate solution standard substance into a 15 mL centrifuge tube, and make up to 10 mL with deionized water to obtain a mixed standard stock solution with a concentration of 1 mg / L.

[0045] Preparation of standard solution: pipette 0.005 mL, 0.01 mL, 0.02 mL, 0.05 mL, 0.10 mL, 0.20 mL, 0.50 mL, and 1.00 mL of 1 mg / L mixed standard solution into 15 mL centrifuge tubes, respectively, and dilute to the scale with water to obtain mixed standard solutions with concentrations of 0.5 µg / L, 1 µg / L, 2 µg / L, 5 µg / L, 10 µg / L, 20 µg / L, 50 µg / L, and 100 µg / L.

[0046] 4 Optimization of arsenic speciation analysis conditions The elution mode of the mobile phase was optimized, and isocratic elution and gradient elution experiments were performed to compare the separation effects.

[0047] 4.1 Isocratic elution First, isocratic elution was adopted, and 10 mM, 20 mM, 30 mM, 50 mM, and 100 mM (NH4)2CO3 were selected as mobile phase B to perform isocratic elution on the standard sample. The obtained chromatogram shows that when the mobile phase concentration is low, the peak elution time will increase. When the mobile phase concentration is high, the peak elution time of each form is close, and the separation effect is poor. Figure 1 .

[0048] 4.2 Gradient elution According to the isocratic elution results in 4.1, the elution mode was adjusted to gradient elution. Two groups of elution modes were selected for comparison. The first group of elution: 5 mM (NH4)2CO3 was selected as mobile phase A and 100 mM (NH4)2CO3 was selected as mobile phase B. Gradient elution was performed according to the volume ratio: 0-6 min, 100%A; 6-10 min, 100%A-0%B; 10-15 min, 0%A--100%B. The second group of elution: 10 mM (NH4)2CO3 was selected as mobile phase A and 100 mM (NH4)2CO3 was selected as mobile phase B. Gradient elution was performed according to the volume ratio: 0-6min, 100%A; 6-10 min, 100%A-0%B; 10-15 min, 0%A--100%B, see Figure 2 .

[0049] According to the test results, the first set of gradient elution has a better separation effect. That is, when 5 mM (NH4)2CO3 is used as mobile phase A and 100 mM (NH4)2CO3 is used as mobile phase B, gradient elution is performed. (For the specific elution procedure, see Fig.10 ) 4.3 Effect of methanol in the mobile phase on arsenic speciation analysis Different concentrations of methanol in the mobile phase will also affect its separation and peak shape. According to the literature and previous experimental results, two concentrations of methanol were set for comparison, 5% and 0%. The experimental results showed that 5% methanol did not damage the separation while improving the response value ( Figure 3 ), so a mobile phase containing 5% methanol was selected in subsequent detection.

[0050] 4.4 Effect of mobile phase pH on arsenic speciation analysis Considering that the pH value of the mobile phase may also affect the peak morphology and elution time of arsenic derivatives, HPLC-ICP-MS detection of 6 arsenic standards was carried out under 3 groups of pH conditions, and the mobile phase pH values ​​were set to 7.5, 9.5, and 10, respectively. The pH steps are as follows: weigh 0.24 g and 4.8 g (NH4)2CO3 respectively, pour them into a 500 mL glass bottle, dissolve them in deionized water to make 500 mL solution, and adjust the pH with ammonia water. When pH = 9.5, the separation is good, see Figure 4 .

[0051] 4.5 Final optimized mobile phase conditions In summary, the optimized mobile phases were: mobile phase A: 5% methanol and 5 mM (NH4)2CO3, and mobile phase B: 5% methanol and 100 mM (NH4)2CO3.

[0052] 5. Optimization of extraction procedures for arsenic derivatives from seafood samples 5.1 Method 1: Extraction with methanol-water (1:1, V / V) solution Approximately 0.1 g of dried and ground marine tissue was added to a 15 mL centrifuge tube, and 10 mL of methanol-water (1:1, V / V) was added. The sample was sonicated in an ultrasonic cleaner for 1 h, shaken in a constant temperature oscillator for 16 h, and centrifuged in a high-speed low-temperature refrigerated centrifuge at 4 ◦ Centrifuge at C-4000×g for 15 min, aspirate the supernatant, extract the precipitate twice, and then adjust the sample supernatant to 10 mL, filter the supernatant with a syringe filter (W / 0.45 μm), and put it on the machine. The standard sample was processed as above.

[0053] Since the concentration of methanol solution was too high, some peaks could not be separated. Therefore, after centrifugation and aspiration of the supernatant, the methanol was blown away with a nitrogen blower (MTN-2800D) and the volume was fixed to 10 mL. The chromatogram obtained had a good separation. Since the recovery rate of As(Ⅲ) was low, the pretreatment method optimization will be continued.

[0054] 5.2 Method 2: Extraction with 5 mM (NH4)2CO3-Methanol mixture (95:5, V / V) The seafood tissue was dried and ground into powder, and 0.1 g was added to a 15 mL centrifuge tube. 5 mL of 5 mM (NH4)2CO3-Methanol mixture (95:5, V / V) was added and shaken to mix. The sample was ultrasonically treated in an ultrasonic cleaner for 30 min (taken out and shaken for 30 s every 10 min), and centrifuged in a high-speed low-temperature refrigerated centrifuge at 4 ◦ C-4000×g centrifugation for 15 min; aspirate the supernatant, extract the precipitate twice, then adjust the sample supernatant to 10 mL, filter the supernatant with a syringe filter (W / 0.45μm), and put it on the machine. The results show that the recovery rates of As(Ⅲ) at three concentrations of high, medium and low are all lower than 60%, and the recovery rates are poor, see Table 4.

[0055] 5.3 Method 3: Microwave-assisted extraction Take 0.1 g of quality control product and add it to a 15 mL centrifuge tube; add 5 mL of 0.5 mM (NH4)2CO3-5%Methanol mixture (95:5, V / V) and shake to mix; digest the sample in a microwave digestion tank [temperature program (50℃ ramp 10 min, hold 10 min, 70℃ ramp 5 min, hold 20 min)]; cool, dilute to 10 mL with deionized water, filter the supernatant with a syringe filter (W / 0.45 μm), and put it on the machine. The results show that the recovery rates of As(Ⅲ) at three concentrations of high, medium and low are all less than 60%, and the recovery rates are poor, as shown in Table 4.

[0056] 5.4 Method 4: Extraction with methanol-water (4:6, V / V) solution (optimal conditions) Weigh about 1 g of sample and add it to a 15 mL centrifuge tube, add 10 mL of methanol-water (4:6, V / V) solution; homogenize at 8000 r / min for 2 min, then ultrasonically extract for 10 min; then transfer to a centrifuge tube, refrigerate centrifuge at 10000 r / min for 15 min; take the supernatant and filter it through a 0.22 μm filter membrane, and analyze the filtrate on the machine. The recovery rates of AsB, DMA, AsC, MMA and As(V) obtained can reach 95% or more, and the recovery rate of As(Ⅲ) is also higher than that of the previous experiment, reaching more than 75%, see Fig.11 .

[0057] 2. Formal experiment of HPLC-ICP-MS detection of arsenic derivatives 6 Optimization results of HPLC-ICP-MS detection conditions 6.1 HPLC-ICP-MS chromatogram of 100 µg / L mixed standard solution of six arsenic species 20 µL of the 100 µg / L mixed standard solution was taken and analyzed in HPLC-ICP-MS. The test was performed according to the instrument conditions in 1.3 to obtain the chromatogram of the standard sample. The results are shown in the figure. All six arsenic forms were well separated within 15 min, and the peaks were symmetrical (see Figure 5 ).

[0058] 6.2 Standard curves for detection of six arsenic derivatives 20 μL of the prepared 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / L solutions were accurately drawn and injected to obtain chromatograms. The retention time was used for qualitative analysis, and the concentration of the target compound in the standard series solution was used as the horizontal axis and the chromatographic peak area was used as the vertical axis to draw a standard curve. The quantitative analysis was performed based on the peak area. The results showed that the linear relationship was good in the range of 0.5 to 100 μg / L. The R 2 All are greater than 0.999. Fig.12 .

[0059] 6.3 Detection limits of six arsenic species by HPLC-ICP-MS 20 μL of the prepared 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / L solutions were accurately pipetted for injection, and the detection limit was a signal-to-noise ratio (S / N) greater than 3, and the quantification limit was a signal-to-noise ratio (S / N) greater than 10. The detection limits of AsB, DMA, As(Ⅲ), AsC, MMA, and As(V) were 0.003, 0.005, 0.006, 0.007, 0.004, and 0.005 μg / L, respectively. The quantification limits were 0.10, 0.16, 0.19, 0.22, 0.12, and 0.17 μg / L, respectively (see Fig.13 ).

[0060] 6.4 Precision of HPLC-ICP-MS for the determination of six arsenic species Accurately pipette 20 μL of the prepared 100 μg / L mixed standard solution of six arsenic forms, inject it 6 times in a row, and test it using the instrument conditions in 1.3 to calculate the precision. It is generally believed that RSD% (relative standard deviation) less than 5% is considered to be good precision. In this experiment, the RSD% of the six derivatives were all less than 5%, which is good precision. Fig.14 .

[0061] 6.5 Results of determination of arsenic derivatives in cell samples After A375 cells were treated according to 2.1, they were tested using the instrument conditions of 1.3. The retention time was used for qualitative analysis. The concentration of the sample was calculated based on the standard curve to obtain the sample spectrum, see Figure 6 .

[0062] 6.6 Detection results of arsenic derivatives in seafood After the seafood samples were processed according to 2.2, they were tested using the instrument conditions of 1.3. The retention time was used for qualitative analysis. The concentration of the samples was calculated based on the standard curve to obtain the sample spectrum, see Figure 7 .

[0063] In summary, a small amount of As(V) was detected in the cells 24 h after administration of ATO. It can be inferred that As(III) was converted into As(V) in human malignant melanoma cells. It is known that the toxicity of As(III) is greater than As(V). It can be considered that this is a detoxification pathway for cells to arsenic. According to the literature, in organisms, for the detoxification of arsenic, As(III) will eventually be converted into less toxic or even non-toxic organic arsenic. Due to the short culture time and the growth characteristics of cells, organic arsenic has not been detected in cells. The arsenic content in seafood samples was also detected. In this test, the detection method was optimized, the concentration and composition of the mobile phase were adjusted, and a new detection method was established. The mobile phase was 5% methanol + 5mmol / L ammonium carbonate mixed solution (A), 5% methanol + 100 mmol / L ammonium carbonate mixed solution (B), and the gradient elution method was used to completely detect six forms of arsenic standards, and the results were good, and the detection limit and precision were good. This experiment also explored that As(III) was converted into As(V) in human malignant melanoma cells within 24 hours, providing a method for the subsequent exploration of arsenic form detection and some evidence for the detoxification mechanism of As(III) in human malignant melanoma cells.

[0064] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for determining arsenic derivatives, characterized in that: The steps include: 1) Pre-treating the sample to be tested containing arsenic derivatives and preparing the sample solution to be tested; 2) The sample solution to be tested is analyzed by high performance liquid chromatography-mass spectrometry; The mobile phases of the elution solution for HPLC elution are: mobile phase A is a mixture of 5% methanol and 5 mmol / L ammonium carbonate, and mobile phase B is a mixture of 5% methanol and 100 mmol / L ammonium carbonate.

2. The method for determining arsenic derivatives according to claim 1, characterized in that: The elution procedure of the high performance liquid chromatography is gradient elution.

3. The method for determining arsenic derivatives according to claim 2, characterized in that: The specific method of the gradient elution comprises: 0-6min, 100% mobile phase A; 6-10min, 100% mobile phase A; 10-15min, 100% mobile phase B; 15-20min, 100% mobile phase B.

4. The method for determining arsenic derivatives according to claim 1, characterized in that: The analysis method is a high performance liquid chromatography-inductively coupled plasma mass spectrometry analysis method.

5. The method for determining arsenic derivatives according to claim 4, characterized in that: The detection conditions of the mass spectrometer are as follows: plasma power is 1550 W, carrier gas is high-purity argon gas; carrier gas flow rate is 1.0 L / min; sampling depth is 5 mm; cooling gas flow rate is 14 L / min; auxiliary gas flow rate is 0.8 L / min; and carrier gas flow rate is 0.9 L / min.

6. The method for determining arsenic derivatives according to any one of claims 1 to 5, characterized in that: The sample to be tested is a cell containing arsenic derivatives; the pretreatment method of the sample to be tested is: adding a lysis solution to the sample to be tested to lyse the cells, shaking, centrifuging and filtering, and taking the filtrate as the sample solution to be tested.

7. The method for determining arsenic derivatives according to claim 5, characterized in that: The amount of the lysate added is 1×10 6 cell / 100 μL RIPA lysis buffer; the shaking time was 5 min; the centrifugal filtration method was to aspirate the cell solution into a centrifuge tube with a 3 kD cut-off membrane, and centrifuge and filter at 4°C and 14000×g for 30 min.

8. The method for determining arsenic derivatives according to any one of claims 1 to 5, characterized in that: The sample to be tested is a marine sample; the pretreatment method of the sample to be tested is: adding the sample to be tested into a methanol-water solution, homogenizing at 8000 r / min for 2 min, and then ultrasonically extracting for 10 min; then transferring to a centrifuge tube, refrigerated centrifuging at 10000 r / min for 15 min; taking the supernatant and filtering it through a 0.22 μm filter membrane, and the obtained filtrate is the sample solution to be tested.

9. The method for determining arsenic derivatives according to claim 8, characterized in that: Each gram of the marine sample is added into 10 mL of a methanol-water solution, wherein the methanol-water solution is a solution with a volume ratio of 4:6.

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