CN108693291B - Method for measuring content of different-form compounds of arsenic and chromium in tobacco and tobacco products - Google Patents
Method for measuring content of different-form compounds of arsenic and chromium in tobacco and tobacco products Download PDFInfo
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Abstract
The invention relates to a method for measuring contents of different morphological compounds of arsenic and chromium in tobacco and tobacco products, belonging to the technical field of heavy metal component measurement in tobacco and tobacco products. The method for measuring the content of the compounds with different forms of arsenic and chromium in the tobacco and the tobacco products comprises the following steps: extracting a sample to be detected, and then adjusting the pH of the extracting solution to 6.0-6.8 to obtain a liquid to be detected; taking a liquid to be detected, and analyzing by adopting a high performance liquid chromatography-mass spectrometry combined method; the high performance liquid chromatography adopts gradient elution. The method for measuring the content of the compounds with different forms of arsenic and chromium in the tobacco and the tobacco products realizes the simultaneous separation and detection of 6 different forms of substances of arsenic and chromium by one-time sample treatment, and the analysis method has the characteristics of high sensitivity, high flux, rapidness, effectiveness and the like.
Description
Technical Field
The invention relates to a method for measuring contents of different morphological compounds of arsenic and chromium in tobacco and tobacco products, belonging to the technical field of heavy metal component measurement in tobacco and tobacco products.
Background
As and Cr elements exist in different forms in nature, wherein Cr exists mainly in the form of trivalent Cr (III) and hexavalent Cr (VI), the toxicity is related to the form, and Cr (III) is necessary for human metabolismTrace elements, oral acute toxicity L D501870mg/kg, and cr (vi) is highly absorbable in humans, readily penetrates cell membranes, has genotoxic and carcinogenic effects, and has oral acute toxicity L D50190mg/kg, inhalation carcinogenesis: IUR (inhalation unit Risk) ═ 12mg/m3. The harm of heavy metal chromium is commonly referred to as the harm of hexavalent chromium to human body. Arsenic can be divided into organic arsenic and inorganic arsenic, As (III), As (V), monomethyl arsenic, dimethyl arsenic and dimethyl arsenic belong to inorganic arsenic, and the toxicity of compounds in different arsenic morphologies is different. The chromium and arsenic which may exist in the tobacco and tobacco products can be leached out along with the action of saliva of the human body and directly enter the human body, thereby causing harm to the human body. Therefore, it is necessary to simultaneously determine arsenic and chromium in tobacco and tobacco products.
In the prior art, a large amount of documents are reported about the analysis of the forms of arsenic and chromium elements, but the documents about the analysis of the forms of the arsenic and chromium elements are concentrated in the fields of food, marine products and environment, and the research documents in the fields of tobacco and snus are less at present, for example, the Chinese invention patent application with the application publication number of CN104833750A discloses a method for separating and measuring chromium elements with different valence states in tobacco and tobacco products, the method adopts a high performance liquid chromatography-inductively coupled plasma mass spectrometer to measure the contents of trivalent chromium and hexavalent chromium in a sample, a mobile phase adopted in the high performance liquid chromatography separation is 0.1 mol/L ammonium nitrate solution with the pH of 7.0, the flow rate is 1.0m L/min, and because the concentration of the mobile phase is too high, the ionic strength in an eluent is too high, the compounds such as methyl arsenic and dimethyl arsenic and the like can hardly remain on an ion chromatographic column, so that the separation and measurement of the compounds with different forms of arsenic and chromium can not be simultaneously measured under the same condition.
Disclosure of Invention
The invention aims to provide a method for measuring contents of different form compounds of arsenic and chromium in tobacco and tobacco products, which can simultaneously measure the contents of the different form compounds of arsenic and chromium.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for measuring the content of different morphological compounds of arsenic and chromium in tobacco and tobacco products comprises the following steps:
extracting a sample to be detected, and then adjusting the pH of the extracting solution to 6.0-6.8 to obtain a liquid to be detected; taking a liquid to be detected, and analyzing by adopting a high performance liquid chromatography-mass spectrometry combined method;
in the analysis process, gradient elution is adopted in high performance liquid chromatography, wherein a mobile phase A adopted in the gradient elution is an ammonium nitrate solution with the pH value of 6.0-6.8 and the concentration of 5-15 mmol/L, a mobile phase B is an ammonium nitrate solution with the pH value of 6.0-6.8 and the concentration of 50-75 mmol/L, the gradient elution program comprises that the mobile phase from 0min to Cmin is 100% A, the Cmin to Dmin is 100% B, the Cmin is selected from any time node between 3.5min and 4.5min, the Cmin is selected from any time node between 18 Dm and 25min, and the flow rate of the mobile phase is 0.5-0.8 m L/min.
The method for measuring the contents of the compounds with different forms of arsenic and chromium in the tobacco and the tobacco products realizes simultaneous separation and detection of 6 substances with different forms of arsenic and chromium (trivalent arsenic, monomethyl arsenic, dimethyl arsenic, pentavalent arsenic, trivalent chromium and hexavalent chromium) by one-time sample treatment, and the analysis method has the characteristics of high sensitivity, high flux, rapidness, effectiveness, wide application range and the like.
The mobile phase A and the mobile phase B are obtained by adjusting the pH value of the ammonium nitrate solution with corresponding concentration. The pH of the mobile phase is adjusted by ammonia water or nitric acid.
The high performance liquid chromatography-mass spectrometry combined method adopts a high performance liquid chromatography-inductively coupled plasma mass spectrometer.
The inductively coupled plasma mass spectrometry conditions comprise emission power of 1320kW, carrier gas flow rate of 1.0-1.1 m L/min, collision gas flow rate of 4.5m L/min, collision gas of helium, peristaltic pump rotation speed of 0.3-0.4 rps, and mass number of analysis75As、52Cr; integration time adoption75As and52cr is 0.5 min; the sampling time is 30 min.
The method for extracting the sample to be detected comprises the following steps: mixing a sample to be detected with hydrochloric acid with the mass fraction of 0.2-0.4%, and then carrying out ultrasonic treatment for 20-50 min.
The volume of hydrochloric acid adopted by each 0.5-1.0 g of sample to be detected is 20-30 m L.
The high performance liquid chromatography adopts an agilent biowax chromatographic column with the chromatographic column of 4 × 25cm, the sample injection amount during the analysis of the high performance liquid chromatography is 10-100 mu L, and the sample injection amount during the sample injection analysis of the high performance liquid chromatography is further preferably 10-50 mu L.
The standard working solution adopted for analysis by the high performance liquid chromatography-mass spectrometry combined method is a mixed standard solution of Cr (III) complex, hexavalent chromium, trivalent arsenic, monomethyl arsenic, dimethyl arsenic and pentavalent arsenic; the Cr (III) complex is obtained by completely complexing a trivalent chromium standard substance by adopting a carboxylic acid complexing agent.
The carboxylic acid complexing agent is Ethylene Diamine Tetraacetic Acid (EDTA).
The method for measuring the content of the compounds with different forms of arsenic and chromium in the tobacco and the tobacco products also comprises the steps of filtering the liquid to be measured by a water-based filter membrane before analyzing the liquid to be measured by adopting a high performance liquid chromatography-mass spectrometry combined method; the pore diameter of the water-based filter membrane is 0.45 mu m.
The method for determining the content of the compounds with different forms of arsenic and chromium in the tobacco and the tobacco products further comprises the steps of analyzing the series of standard solutions by adopting a high performance liquid chromatography-mass spectrometry method, and then drawing a standard curve by taking the concentration and the peak area of the series of standard solutions as vertical and horizontal coordinates respectively. The conditions for analyzing the series of standard solutions by adopting the high performance liquid chromatography-mass spectrometry method are the same as the conditions for analyzing the liquid to be detected by adopting the high performance liquid chromatography-mass spectrometry method. And (4) quantifying according to a standard curve by using the peak areas of trivalent arsenic, monomethylarsenic, dimethylarsine, pentavalent arsenic, trivalent chromium and hexavalent chromium in the solution to be detected on an instrument.
Drawings
FIG. 1 shows the extracted ions (As) of trivalent arsenic, monomethylarsenic, dimethylarsine and pentavalent arsenic in the mixed standard solution of example 175) A chromatogram;
FIG. 2 shows the extracted ions (Cr) of trivalent chromium and hexavalent chromium in the mixed standard solution of example 152) And (4) chromatograms.
Detailed Description
The technical solution of the present invention will be further described below with reference to specific embodiments.
In the specific embodiment, the chromatographic column in the high performance liquid chromatography-inductively coupled plasma mass spectrometer is agilent biomax (4 × 25cm), and during analysis, the inductively coupled plasma mass spectrometry conditions are that the emission power is 1320kW, the carrier gas flow rate is 1.10m L/min (Ar), the collision gas flow rate is 4.5m L/min (He), the peristaltic pump rotation speed is 0.3rps, the integration time is 0.5min, the sampling time is 15min, and the analysis mass number:75As、52Cr；
the mixed standard solution used in each example was prepared by a method comprising the following steps:
i) dissolving a trivalent chromium standard substance in water to prepare a solution with a trivalent chromium ion concentration of 100 mu g/L, then placing the solution in a thermostatic water bath at 80 ℃, heating to 80 ℃, adding 2 mmol/L of EDTA solution, and reacting for 40min to fully complex trivalent chromium to obtain a Cr (III) complex solution;
ii) respectively preparing a trivalent arsenic standard solution, a monomethyl arsenic standard solution, a dimethyl arsenic standard solution, a pentavalent arsenic standard solution and a hexavalent chromium standard solution with certain concentrations, and then respectively mixing a proper amount of Cr (III) complex solution, the trivalent arsenic standard solution, the monomethyl arsenic standard solution, the dimethyl arsenic standard solution, the pentavalent arsenic standard solution and the hexavalent chromium standard solution to obtain a mixed standard solution; diluting the obtained mixed standard solution to obtain a series of standard working solutions with a certain concentration gradient, wherein the concentration gradient of each standard working solution is shown in table 1.
TABLE 1 gradient of concentrations of the respective standard working solutions (unit: μ g/L)
In the preparation of the mobile phase a and the mobile phase B in each embodiment, ammonium nitrate is firstly dissolved in water to prepare ammonium nitrate solution with corresponding concentration, and then ammonia water or nitric acid is respectively adopted to adjust the pH to be in a corresponding range.
Example 1
The method for measuring the content of the arsenic and chromium compounds in different forms in the tobacco and the tobacco products in the embodiment is to measure the content of the arsenic and chromium compounds in different forms in bulk buccal tobacco, and comprises the following steps:
1) weighing 0.5g of ground bulk buccal tobacco sample, putting the ground bulk buccal tobacco sample into a conical flask, adding 25m L hydrochloric acid with the mass fraction of 0.2%, putting the ground tobacco sample into ultrasonic waves after the tobacco sample is completely submerged, performing ultrasonic extraction for 30min, and performing solid-liquid separation to obtain an extract liquid, wherein the ultrasonic wave power is 300W, and the frequency is 40 KHz;
2) filtering the extract obtained in the step 1) with a 0.45-micron aqueous filter membrane to obtain a filtrate, adjusting the pH of the filtrate to 6.0-6.8 by using ammonia water, and taking the filtrate after the pH is adjusted by 1m L as a solution to be detected and placing the solution in a chromatographic bottle;
3) taking the liquid to be detected 1m L obtained in the step 2) and analyzing by adopting a high performance liquid chromatography-inductively coupled plasma mass spectrometer;
the chromatographic conditions used for the analysis were 15 mmol/L ammonium nitrate (pH 6.5) for mobile phase A and 50 mmol/L ammonium nitrate (pH 6.5) for mobile phase B, a flow rate of 0.6m L/min, a mobile phase gradient program of t 0.0min, 100% A, t 4.0min, 100% B, t25 min, 100% A, t 30min, 100% A, a column oven at room temperature, a sample throughput of 50 μ L;
4) under the condition of analyzing by adopting a high performance liquid chromatography-inductively coupled plasma mass spectrometer in the step 3), drawing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate; extracting ions (As) of trivalent arsenic, monomethylarsenic, dimethylarsine and pentavalent arsenic in mixed standard solution75) The chromatogram is shown in FIG. 1; extracting ion (Cr) of trivalent chromium and hexavalent chromium in mixed standard solution52) The chromatogram is shown in FIG. 2;
the peak area size measured in step 3) was quantified and the results of the analysis are shown in Table 2.
TABLE 2 measurement results of As and Cr in bulk buccal tobacco samples
Example 2
The method for measuring the content of the compounds with different forms of arsenic and chromium in the tobacco and the tobacco products in the embodiment is to measure the content of the compounds with different forms of arsenic and chromium in the tobacco, and comprises the following steps:
1) weighing 0.5g of ground tobacco sample, putting the tobacco sample into a conical flask, adding 25m L hydrochloric acid with the concentration of 0.2%, putting the sample into ultrasonic waves after the solution is completely immersed into the sample, performing ultrasonic extraction for 30min, and performing solid-liquid separation to obtain an extract liquid, wherein the ultrasonic power is 300W, and the frequency is 40 KHz;
2) filtering the extract obtained in the step 1) with a 0.45-micron water-phase filter membrane to obtain a filtrate, adjusting the pH of the filtrate to 6.0-6.8 by using ammonia water, and taking the filtrate obtained after the pH is adjusted by 1m L as a solution to be detected and placing the solution in a chromatographic bottle;
3) analyzing a liquid to be detected by a high performance liquid chromatography-inductively coupled plasma mass spectrometer under the chromatographic conditions that a mobile phase A is 15 mmol/L ammonium nitrate (pH is 6.5), a mobile phase B is 50 mmol/L ammonium nitrate (pH is 6.5), the flow rate is 0.6m L/min, the mobile phase gradient program is that t is 0.0min, 100 percent A, t is 4.0min, 100 percent B, t is 25min, 100 percent A, t is 30min and 100 percent A, the column temperature box is at room temperature, and the sample introduction amount is 50 mu L;
4) under the condition of analyzing by adopting a high performance liquid chromatography-inductively coupled plasma mass spectrometer in the step 3), drawing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate; the peak area size measured in step 3) was quantified and the results of the analysis are shown in Table 3.
TABLE 3 measurement results of As and Cr elements in tobacco
| Test object | Measured value (μ g/kg) |
| As(Ⅲ) | 11.5 |
| As(Ⅴ) | 40.8 |
| Monomethyl arsenic | 7.8 |
| Dimethyl arsenic | 10.4 |
| Cr(Ⅲ) | 36.4 |
| Cr(Ⅵ) | 2.7 |
According to the method for measuring the content of the compounds with different forms of arsenic and chromium in the tobaccos and tobacco products in example 2, the repeatability and the recovery rate of the method are examined by using the samples in example 2, the sample injection is repeated for 10 times at the lowest point of the standard curve, the standard deviation of the 10 sample injection results is used as the detection limit of the method, and the results of the repeatability, the recovery rate and the detection limit are shown in Table 4.
TABLE 4 repeatability, recovery and detection limits
Note that the variation coefficient and recovery rate in Table 4 were in%, the detection limit in. mu.g/L and the other values in. mu.g/kg.
Example 3
The method for determining the content of the compounds with different forms of arsenic and chromium in the tobacco and the tobacco products comprises the following steps:
1) weighing 1g of buccal tobacco sample, putting the buccal tobacco sample into a conical flask, adding 50m L hydrochloric acid with the concentration of 0.4%, putting the buccal tobacco sample into ultrasonic waves after the solution completely immerses the sample, performing ultrasonic extraction for 30min, and performing solid-liquid separation to obtain an extract liquid, wherein the ultrasonic power is 300W, and the frequency is 40 KHz;
2) filtering the extract obtained in the step 1) with a 0.22-micron water-phase filter membrane to obtain a filtrate, adjusting the pH of the filtrate to 6.0-6.8 by using ammonia water, and taking the filtrate obtained after the pH is adjusted by 1m L as a solution to be detected and placing the solution in a chromatographic bottle;
3) analyzing the liquid to be detected by a high performance liquid chromatography-inductively coupled plasma mass spectrometer under the chromatographic conditions of 10 mmol/L ammonium nitrate (pH6.8) as a mobile phase A and 60 mmol/L ammonium nitrate (pH6.0) as a mobile phase B, 0.6m L/min as a flow rate, 0.0min as a mobile phase gradient program, 100% A, 4.0min as a mobile phase gradient program, 100% B, 20min as a mobile phase B, 100% A, 30min as a mobile phase gradient program and 100% A as a mobile phase gradient program, and 10 mu L as a column incubator at room temperature;
4) under the condition of analyzing by adopting a high performance liquid chromatography-inductively coupled plasma mass spectrometer in the step 3), drawing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate; quantifying according to the peak area size measured in the step 3).
Example 4
The method for determining the content of the compounds with different forms of arsenic and chromium in the tobacco and the tobacco products comprises the following steps:
1) weighing 1.0g of tobacco sample, putting the tobacco sample into a conical flask, adding 50m L hydrochloric acid with the concentration of 0.4%, putting the tobacco sample into ultrasonic waves after the solution completely immerses the sample, performing ultrasonic extraction for 30min, and performing solid-liquid separation to obtain an extract liquid, wherein the ultrasonic power is 300W and the frequency is 40 KHz;
2) filtering the extract obtained in the step 1) with a 0.45-micron water-phase filter membrane to obtain a filtrate, adjusting the pH of the filtrate to 6.0-6.8 by using ammonia water, and taking the filtrate obtained after the pH is adjusted by 1m L as a solution to be detected and placing the solution in a chromatographic bottle;
3) analyzing the liquid to be detected by a high performance liquid chromatography-inductively coupled plasma mass spectrometer under the chromatographic conditions of 5 mmol/L ammonium nitrate (pH6.0) as a mobile phase A and 75 mmol/L ammonium nitrate (pH6.8) as a mobile phase B, 0.6m L/min as a flow rate, 0.0min as a mobile phase gradient program, 100% A, 3.5min as a mobile phase gradient program, 100% B, 18min as a mobile phase B, 100% A, 30min as a mobile phase gradient program and 100% A as a mobile phase gradient program, and 10 mu L as a column incubator at room temperature;
4) under the condition of analyzing by adopting a high performance liquid chromatography-inductively coupled plasma mass spectrometer in the step 3), drawing a standard curve by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate; quantifying according to the peak area size measured in the step 3).
Claims (7)
1. A method for measuring the content of different morphological compounds of arsenic and chromium in tobacco and tobacco products is characterized in that: the method comprises the following steps:
extracting a sample to be detected, and then adjusting the pH of the extracting solution to 6.0-6.8 to obtain a liquid to be detected; taking a liquid to be detected, and analyzing by adopting a high performance liquid chromatography-mass spectrometry combined method;
in the analysis process, the chromatographic column adopted by the high performance liquid chromatography is an agilent biowax chromatographic column of 4 × 25 cm;
performing high performance liquid chromatography by gradient elution;
the mobile phase A adopted by the gradient elution is an ammonium nitrate solution with the pH = 6.0-6.8 and the concentration of 5-15 mmol/L, and the mobile phase B is an ammonium nitrate solution with the pH = 6.0-6.8 and the concentration of 50-75 mmol/L;
the gradient elution procedure comprises that the mobile phase of 0-Cmin is 100 percent A, the mobile phase of Cmin-Dmin is 100 percent B, the Cmin is selected from any time node between 3.5min and 4.5min, the Dmin is selected from any time node between 18min and 25min, and the flow rate of the mobile phase is 0.5-0.8 m L/min;
the compounds of arsenic and chromium in different forms comprise trivalent arsenic, monomethyl arsenic, dimethyl arsenic, pentavalent arsenic, trivalent chromium and hexavalent chromium.
2. The method for determining the content of arsenic and chromium compounds in different forms in tobacco and tobacco products according to claim 1, wherein: the high performance liquid chromatography-mass spectrometry combined method adopts a high performance liquid chromatography-inductively coupled plasma mass spectrometer.
3. The method according to claim 2, wherein the inductively coupled plasma mass spectrometry is performed at an emission power of 1320kW, the carrier gas flow rate of Ar is 1.0-1.1 m L/min, the collision gas flow rate of Ar is 4.5m L/min, the collision gas is helium, the rotation speed of the peristaltic pump is 0.3-0.4 rps, and the analysis mass number is 0.3-0.4 rps75As、52Cr; integration time adoption75As and52cr is 0.5 min; the sampling time is 30 min.
4. The method for determining the content of arsenic and chromium compounds in different forms in tobacco and tobacco products according to claim 1, wherein: the method for extracting the sample to be detected comprises the following steps: mixing a sample to be detected with hydrochloric acid with the mass fraction of 0.2-0.4%, and then carrying out ultrasonic treatment for 20-50 min.
5. The method for determining the content of the compounds with different forms of arsenic and chromium in the tobaccos and the tobacco products as claimed in claim 4, wherein the volume of the hydrochloric acid adopted by each 0.5-1.0 g of the sample to be tested is 20-30 m L.
6. The method for determining the content of arsenic and chromium compounds in different forms in tobacco and tobacco products according to claim 1, wherein: the standard working solution adopted for analysis by the high performance liquid chromatography-mass spectrometry combined method is a mixed standard solution of Cr (III) complex, hexavalent chromium, trivalent arsenic, monomethyl arsenic, dimethyl arsenic and pentavalent arsenic; the Cr (III) complex is obtained by completely complexing a trivalent chromium standard substance by adopting a carboxylic acid complexing agent.
7. The method for determining the content of arsenic and chromium compounds in different forms in tobacco and tobacco products according to claim 1, wherein: the method also comprises the steps of filtering the solution to be detected by adopting a water-based filter membrane before the solution to be detected is analyzed by adopting a high performance liquid chromatography-mass spectrometry combined method; the pore diameter of the water-based filter membrane is 0.45 mu m.
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