CN115096986A - Method for detecting methyl mercury isotope of biological sample with high sensitivity - Google Patents

Abstract

The invention discloses a method for detecting methyl mercury isotopes of a biological sample with high sensitivity, which directly and accurately measures the methyl mercury content of the biological sample, fully extracts the methyl mercury without inputting exogenous mercury, ensures that the ratio of the methyl mercury isotopes extracted by the method is consistent with that of an actual sample, increases the analysis precision of the methyl mercury isotopes of the biological sample by adopting an off-line pretreatment method, and improves the operability of the methyl mercury isotope measuring process, and comprises the following steps: the method comprises the steps of preparing before an experiment, pretreating a sample, detecting the recovery rate and purity and detecting mercury isotopes, wherein reagents such as toluene, copper sulfate, sodium bromide and the like are fully vibrated and mixed with a biological sample, so that methyl mercury in the sample is extracted into an organic phase, then, the methyl mercury is extracted from the organic phase by using a sodium thiosulfate solution, and the methyl mercury content of the sodium thiosulfate solution is determined by analyzing the methyl mercury content and the methyl mercury isotope characteristics of the sample.

Description

A high-sensitivity method for the detection of methylmercury isotopes in biological samples

technical field

The invention relates to the technical field of detection, in particular to a method for detecting methylmercury isotopes of biological samples with high sensitivity.

Background technique

Mercury is the only heavy metal that can exist in the environment in the form of gaseous elemental substances. It has the characteristics of persistence, long-distance transport and bioaccumulation, and is one of the most toxic heavy metals. Mercury exists mainly in inorganic and organic forms in nature, and its occurrence form is closely related to environmental behavior, physiological toxicity and bioavailability. Among the common mercury compounds, methylmercury is the most toxic. The absorption efficiency of methylmercury in the human small intestine is about 95%, and it is easy to pass through the placental barrier and the blood-brain barrier, thereby causing serious harm to the nervous system. Therefore, identifying the migration, transformation process and source of methylmercury is crucial for clarifying the toxicity and physiological hazards of mercury.

At present, measurement methods such as atomic spectroscopy, gas chromatography, and inductively coupled plasma-mass spectrometry have been used to measure methylmercury levels in biological samples of different matrices closely related to humans, such as vegetables, rice, fish, and hair. With the development and application of multi-receiver plasma mass spectrometer (MC-ICP-MS) and the progress of sample preparation technology, non-traditional stable isotopes have been widely used in environmental pollutant traceability and process tracing. It has become an important research method in international earth science and environmental science. Total mercury isotopes have been widely used in the tracer of mercury exposure sources and metabolic processes in the population, but there is still no method for high-precision offline measurement of methylmercury isotopes in different biological samples. Therefore, a new type of methylmercury isotope was developed. Pretreatment techniques are of great significance for identifying the biogeochemical properties of mercury.

SUMMARY OF THE INVENTION

In view of the above problems, the present invention provides a high-sensitivity detection of methylmercury isotopes for biological samples.

The technical scheme of the present invention is: a high-sensitivity method for detecting methylmercury isotopes of biological samples, comprising the following steps:

O1: Preparation before testing

Testing consumables and equipment: toluene, sulfuric acid, nitric acid, hydrochloric acid, sodium bromide, anhydrous copper sulfate, sodium thiosulfate, potassium bromate, potassium bromide, 200mL borosilicate glass bottle, 1000mL borosilicate glass bottle, 15mL polypropylene centrifuge tube , 50mL polypropylene centrifuge tubes, horizontal shakers, muffle furnaces, centrifuges, vortexers, high-speed centrifuges and refrigerators;

Soak the glassware required for detection in 20% HNO ₃ solution for more than 24 hours, rinse with deionized water for more than three times after completion, and then use a muffle furnace to bake it at 500 °C;

Reagent preparation:

NaBr solution: dissolve 155g NaBr with 390mL of H ₂ O, then add 110 mL of H ₂ SO ₄ to prepare a w/w NaBr solution with a concentration of 30%;

CuSO ₄ solution: weigh 25g of anhydrous CuSO ₄ into 1000mL of H ₂ O to prepare a w/w CuSO ₄ solution with a concentration of 2.5%;

Na ₂ S ₂ O ₃ solution: weigh 800 mg of Na ₂ S ₂ O ₃ into 1000 mL of H ₂ O, and prepare a Na ₂ S ₂ O ₃ solution with a concentration of 0.005mol/L;

BrCl solution: accurately weigh 0.76g KBrO ₃ and 0.54g KBr into a glass bottle, burn in a muffle furnace at 250°C for 12h, add 10mL of H ₂ O and 40mL of HCl after cooling to prepare 0.2mol/L of BrCl;

NH ₂ OHHCl solution: Weigh 25 g of NH ₂ OHHCl and dissolve it in 1000 mL of H ₂ O in a glass bottle to prepare a 0.36 mol/L NH ₂ OHHCl solution.

O2: Sample processing procedure

Accurately weigh 0.1~1g of biological sample into a 50mL centrifuge tube, add 5mL of 30%w/w acidic NaBr solution and 10mL of 2.5%w/w _CuSO4 solution, and weigh the mass of the entire centrifuge tube, denoted as m1;

Add 10 mL of toluene solution to the above centrifuge tube, weigh the mass of the entire centrifuge tube, record it as m2;

Using a horizontal shaker, the above solution was shaken at a speed of 420rpm for 1.5h, and then centrifuged at 3000rpm for 15min with a centrifuge until the toluene phase was transparent;

Recover the toluene phase from a 50mL centrifuge tube into a 15mL centrifuge tube, and accurately weigh the recovered toluene phase mass, denoted as m3;

Add 4 mL of 0.005mol/L Na ₂ S ₂ O ₃ solution to a 15 mL centrifuge tube, vortex for 3 min, and then shake for 30 min;

Centrifuge the 15mL centrifuge tube for 30min at 4000rpm with a centrifuge, recover the sodium thiosulfate phase as much as possible into a new centrifuge tube, and store it in the refrigerator for testing. This solution is recorded as S1;

Accurately weigh 0.1~1g of biological sample into a 50mL centrifuge tube, add 5ml of HNO ₃ with a concentration of 25% to the centrifuge tube, digest at 70-75°C for 12 hours, and after the digestion is completed, use 60°C ultrapure water to determine To be tested, the solution is recorded as S2.

O3: Recovery and Purity Testing

Draw an appropriate amount of S1 solution, dilute it with 10% HNO ₃ to an appropriate concentration, gradually add 0.05 mL of 0.2 mol/L BrCl solution until the solution is slightly yellow, put it in the refrigerator for 12 hours to be tested, the solution is recorded as S3;

Draw an appropriate amount of S1 solution, dilute it with H2O to an appropriate concentration, put it in the refrigerator for testing, and record the solution as S4;

Subsequently, the methylmercury concentration of S2 solution was measured by atomic gas chromatography, and recorded as C1; the total mercury concentration of S3 solution was measured by cold atomic fluorescence method, and it was recorded as C2; the methylmercury concentration of S4 solution was measured by atomic gas chromatography, and it was recorded as C3 ;

The recovery rate is expressed as C3/C1, and the purity is expressed as C3/C2. Only when the sample recovery rate and purity are higher than 90% are used for methylmercury isotope analysis, so as to ensure that the methylmercury of the biological sample is completely extracted during the detection process, and there is no inorganic Introduction of mercury.

O4: Mercury isotope detection

Gradually add 0.05 mL of 0.2 mol/L BrCl solution to the S1 solution with a recovery rate and purity higher than 90% until the solution is slightly yellow, put it in the refrigerator for 12 hours to be tested, before the sample test, use a concentration of 20% v/v anti-aqua regia was diluted to an appropriate concentration, and excess BrCl was reduced with NH2OHHCl solution for the analysis of methylmercury isotopic characteristics by multi-receiver inductively coupled plasma mass spectrometry (MC-ICPMS).

Further, the purity of the NaBr is 99.5%.

Further, it is characterized in that the purity of the anhydrous CuSO ₄ and the purity of NH ₂ OHHCl are both higher than 99%.

On the basis of the foregoing scheme, it should also be noted that the purity of the Na ₂ S ₂ O ₃ is 98%.

On the basis of the foregoing scheme, it should be further explained that the anti-aqua regia is prepared by HNO ₃ : HCl in a ratio of 3:1.

The beneficial effects of the present invention are:

1. The method for detecting methylmercury isotopes in biological samples directly and accurately measures the methylmercury content of biological samples;

2. In the whole experimental process of this method, methylmercury is fully extracted, and there is no input of exogenous mercury, which ensures that the isotope ratio of methylmercury extracted by this method is consistent with the actual sample;

3. The method adopts an offline pretreatment method, which increases the analytical precision of methylmercury isotopes of biological samples and improves the operability of the methylmercury isotope measurement process.

Detailed ways

The embodiments of the present invention will be further described below.

Example:

A high-sensitivity method for the detection of methylmercury isotopes in biological samples

D1: Preparation before testing

Testing consumables and equipment: toluene, sulfuric acid, nitric acid, hydrochloric acid, sodium bromide, anhydrous copper sulfate, sodium thiosulfate, potassium bromate, potassium bromide, 200mL borosilicate glass bottle, 1000mL borosilicate glass bottle, 15mL polypropylene centrifuge tube , 50mL polypropylene centrifuge tubes, horizontal shakers, muffle furnaces, centrifuges, vortexers, high-speed centrifuges and refrigerators;

Soak the glassware required for detection in 20% HNO3 solution for more than 24 hours, rinse with deionized water for more than three times after completion, and then use a muffle furnace to bake it at 500 °C;

Reagent preparation:

NaBr solution: dissolve 155g of NaBr with 390mL of _H2O , then add 110mL of _H2SO4 to prepare a w/w NaBr solution with a concentration of 30%, wherein the purity of NaBr used is 99.5%;

CuSO ₄ solution: weigh 25g of anhydrous CuSO ₄ into 1000mL of H ₂ O to prepare a w/w CuSO ₄ solution with a concentration of 2.5%, the purity of the anhydrous CuSO ₄ used is higher than 99%;

Na ₂ S ₂ O ₃ solution: Weigh 800 mg of Na ₂ S ₂ O ₃ into 1000 mL of H ₂ O to prepare a Na ₂ S ₂ O ₃ solution with a concentration of 0.005 mol/L, wherein the used Na ₂ S ₂ O ₃ is 98% pure;

BrCl solution: accurately weigh 0.76g KBrO ₃ and 0.54g KBr into a glass bottle, burn in a muffle furnace at 250°C for 12h, add 10mL of H ₂ O and 40mL of HCl after cooling to prepare 0.2mol/L of BrCl;

NH ₂ OHHCl solution: Weigh 25 g of NH ₂ OHHCl and dissolve it in 1000 mL of H ₂ O in a glass bottle to prepare a 0.36 mol/L NH ₂ OHHCl solution. The purity of the used NH ₂ OHHCl is higher than 99%.

D2: Sample total mercury content test:

The total mercury content of biological samples was directly measured by DMA80 mercury measuring instrument.

D3: Sample processing procedure

Accurately weigh 0.1~1g of biological sample into a 50mL centrifuge tube, add 5mL of 30%w/w acidic NaBr solution and 10mL of 2.5%w/w _CuSO4 solution, and weigh the mass of the entire centrifuge tube, denoted as m1;

Add 10 mL of toluene solution to the above centrifuge tube, weigh the mass of the entire centrifuge tube, record it as m2;

Using a horizontal shaker, the above solution was shaken at a speed of 420rpm for 1.5h, and then centrifuged at 3000rpm for 15min with a centrifuge until the toluene phase was transparent;

Recover the toluene phase from a 50mL centrifuge tube into a 15mL centrifuge tube, and accurately weigh the recovered toluene phase mass, denoted as m3;

Add 4 mL of 0.005mol/L Na ₂ S ₂ O ₃ solution to a 15 mL centrifuge tube, vortex for 3 min, and then shake for 30 min;

Centrifuge the 15mL centrifuge tube for 30min at 4000rpm with a centrifuge, recover the sodium thiosulfate phase as much as possible into a new centrifuge tube, and store it in the refrigerator for testing. This solution is recorded as S1;

Accurately weigh 0.1~1g of biological sample into a 50mL centrifuge tube, add 5ml of HNO ₃ with a concentration of 25% to the centrifuge tube, digest at 70-75°C for 12 hours, and after the digestion is completed, use 60°C ultrapure water to determine To be tested, the solution is recorded as S2.

D4: Recovery and Purity Testing

Draw an appropriate amount of S1 solution, dilute it with 10% HNO ₃ to an appropriate concentration, gradually add 0.05 mL of 0.2 mol/L BrCl solution until the solution is slightly yellow, put it in the refrigerator for 12 hours to be tested, the solution is recorded as S3;

Draw an appropriate amount of S1 solution, dilute it with H2O to an appropriate concentration, put it in the refrigerator for testing, and record the solution as S4;

Subsequently, the methylmercury concentration of S2 solution was measured by atomic gas chromatography, and recorded as C1; the total mercury concentration of S3 solution was measured by cold atomic fluorescence method, and it was recorded as C2; the methylmercury concentration of S4 solution was measured by atomic gas chromatography, and it was recorded as C3 ;

The recovery rate is expressed as C3/C1, and the purity is expressed as C3/C2. Only when the sample recovery rate and purity are higher than 90% are used for methylmercury isotope analysis, so as to ensure that the methylmercury of the biological sample is completely extracted during the detection process, and there is no inorganic Introduction of mercury.

D5: Mercury isotope detection

Gradually add 0.05 mL of 0.2 mol/L BrCl solution to the S1 solution with a recovery rate and purity higher than 90% until the solution is slightly yellow, put it in the refrigerator for 12 hours to be tested, before the sample test, use a concentration of 20% v/v anti-aqua regia was diluted to an appropriate concentration, and excess BrCl was reduced with NH2OHHCl solution for the analysis of methylmercury isotopic characteristics by multi-receiver inductively coupled plasma mass spectrometer (MC-ICPMS). Anti-aqua regia was HNO ₃ : HCl according to 3 : 1 ratio configuration.

Specifically, the test process of total mercury content in D2 sample is to estimate the sample weight required by this method, and the standard curve, system blank, standard material and random sample parallel test are used for quality control, and the detection limit of total mercury in the sample is 0.05ng/ g; After D1 treatment, the standard curve, system blank, method blank, standard addition recovery, standard material and random sample parallel testing were used for quality control in the D4 recovery and purity testing process. The detection limit of sample methylmercury was 0.06ng/g.

The above-mentioned embodiments only represent specific embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention.

Claims (5)

1. A method for detecting a methyl mercury isotope of a biological sample with high sensitivity comprises the following steps:

o1: preparation before detection

Detecting consumables and equipment: toluene, sulfuric acid, nitric acid, hydrochloric acid, sodium bromide, anhydrous copper sulfate, sodium thiosulfate, potassium bromate, potassium bromide, 200mL borosilicate glass bottles, 1000mL borosilicate glass bottles, 15mL polypropylene centrifuge tubes, 50mL polypropylene centrifuge tubes, horizontal oscillators, muffle furnaces, centrifuges, vortexers, high-speed centrifuges and refrigerators;

soaking glassware required for detection in 20% HNO ₃ Washing the solution for more than three times by using deionized water after the solution is dissolved for more than 24 hours, and then completely baking the solution by using a muffle furnace at the temperature of 500 ℃;

preparing a reagent:

NaBr solution: using 390mL of H ₂ O dissolved 155gNaBr, then added 110mLH ₂ SO ₄ Preparing a w/wNaBr solution with the concentration of 30 percent;

CuSO ₄ solution: weigh 25g of anhydrous CuSO ₄ To 1000mL of H2O, formulated as 2.5% w/w CuSO ₄ A solution;

Na ₂ S2O ₃ solution: weigh 800mg of Na ₂ S ₂ O ₃ To 1000mLH ₂ In O, Na is prepared with a concentration of 0.005mol/L ₂ S ₂ O ₃ A solution;

BrCl solution: accurately weighing 0.76g KBrO ₃ And 0.54g of KBr in a glass bottle, firing the mixture at 250 ℃ for 12 hours in a muffle furnace, cooling the mixture, and adding 10mL of H ₂ O and 40mL of HCl are prepared into 0.2mol/L BrCl;

NH ₂ OHHCl solution: 25g of NH were weighed ₂ OHCl in 1000mL of H ₂ O in a glass bottle to prepare 0.36mol/L NH ₂ OHHCl solution.

O2: sample processing procedure

Accurately weighing 0.1-1 g of biological sample into a 50mL centrifuge tube, adding 5mL of 30% w/w acidic NaBr solution and 10mL of 2.5% w/w CuSO ₄ Weighing the mass of the whole centrifuge tube, and marking as m 1;

adding 10mL of toluene solution into the centrifuge tube, and weighing the mass of the whole centrifuge tube, wherein the mass is recorded as m 2;

shaking the solution at 420rpm for 1.5h by using a horizontal oscillator, and then centrifuging the solution at 3000rpm by using a centrifuge for 15min until the toluene phase is transparent;

recovering a toluene phase from a 50mL centrifuge tube into a 15mL centrifuge tube, and accurately weighing the mass of the recovered toluene phase, wherein the mass is recorded as m 3;

4mL of a solution having a concentration of 0.005mol/LNa was added to a 15mL centrifuge tube ₂ S ₂ O ₃ The solution is firstly vortexed for 3min and then shaken for 30 min;

centrifuging a 15mL centrifuge tube for 30min at 4000rpm of a centrifuge, recovering a sodium thiosulfate phase into a new centrifuge tube as far as possible, storing the sodium thiosulfate phase in a refrigerator to be tested, and recording the solution as S1;

accurately weighing 0.1-1 g of biological sample into a 50mL centrifuge tube, and adding 5mL of 25% HNO into the centrifuge tube ₃ Digesting for 12h at the temperature of 70-75 ℃, after digestion, fixing the volume to be measured by using ultrapure water at 60 ℃, and recording the solution as S2.

O3: recovery and purity measurements

Sucking appropriate amount of S1 solution, and adding 10% HNO ₃ Diluting to a proper concentration, gradually adding 0.05mL of 0.2mol/L BrCl solution until the solution is yellowish, putting the solution into a refrigerator for 12 hours to be tested, and marking the solution as S3;

sucking appropriate amount of S1 solution, and adding H ₂ Diluting O to a proper concentration, putting the solution into a refrigerator to be tested, and marking the solution as S4;

then measuring the concentration of the methylmercury in the S2 solution by using atomic gas chromatography, and recording as C1; measuring the total mercury concentration of the S3 solution by using a cold atom fluorescence method, and recording as C2; measuring the concentration of the methyl mercury in the S4 solution by using atomic gas chromatography, and recording as C3;

the recovery rate is represented by C3/C1, the purity is represented by C3/C2, and the sample recovery rate and purity are ensured to be higher than 90% for methyl mercury isotope analysis, so that the biological sample methyl mercury is completely extracted in the detection process, and no inorganic mercury is introduced.

O4: mercury isotope detection

Gradually adding 0.05mL of 0.2mol/L BrCl solution into S1 solution with recovery rate and purity higher than 90% until the solution is yellowish, placing in a refrigerator for 12h to be tested, diluting with 20% v/v of reverse aqua regia to appropriate concentration before testing, and using NH ₂ And reducing excessive BrCl by the OHCl solution to prepare a multi-receiving inductively coupled plasma mass spectrometer for analyzing the methyl mercury isotope characteristics, wherein the multi-receiving inductively coupled plasma mass spectrometer is MC-ICPMS.

2. The method for detecting methylmercury isotopes of biological samples with high sensitivity according to claim 1, wherein the purity of NaBr is 99.5%.

3. The method of claim 1, wherein the biological sample is detected with high sensitivityMethod for the production of methyl mercury isotopes, characterised in that said anhydrous CuSO is obtained ₄ Purity and NH of ₂ The purity of OHCl is higher than 99%.

4. The method for detecting methylmercury isotope in biological samples with high sensitivity according to claim 1, wherein the Na is ₂ S2O ₃ The purity of (2) was 98%.

5. The method for detecting methylmercury isotope in biological samples with high sensitivity according to claim 1, wherein the reverse aqua regia is HNO ₃ : HCl as 3: 1, is prepared according to the proportion of 1.