阅读说明：本技术 一种沉积物中***药物残留的高通量高精度检测方法 (High-flux high-precision detection method for human and veterinary drug residues in sediment ) 是由 俞慎 洪兵 周敏 于 2018-07-03 设计创作，主要内容包括：本发明专利针对人兽药物多组分残留高通量痕量检测技术的不足,提供了一种沉积物中人兽药物残留的高通量高精度检测方法,所述方法可通过沉积物单一处理流程同时进行多类别多组分人兽药物残留的高通量痕量检测。所述发明专利检测技术流程包括沉积物样采集及保存,样品前处理,固相萃取富集,氮吹浓缩,定容,高效液相色谱串联质谱仪测定,数据分析和方法适用范围。本方法具备前处理方法简便高效、单批次检测药物组分多、所测药物精度高和不同pH的萃取剂萃取共同药物化合物的检测结果可进行交叉校验等特点,极大程度地克服了不同组分多次测定带来的分析误差,提高了沉积物样品中痕量残留人兽药物化合物不同组分间的可对比性和方法稳定性。随着可获取药物化合物标准物质的增加,可进一步扩展适用药物化合物范围。(The invention provides a high-flux high-precision detection method for human and animal medicine residues in sediments, aiming at the defects of a high-flux trace detection technology for human and animal medicine multi-component residues. The invention discloses a detection technical process which comprises the steps of sediment sample collection and storage, sample pretreatment, solid phase extraction and enrichment, nitrogen blowing and concentration, constant volume, high performance liquid chromatography tandem mass spectrometry determination, data analysis and method application range. The method has the characteristics of simple and efficient pretreatment method, high single-batch detection of multiple drug components, high accuracy of the detected drugs, capability of cross-checking the detection results of the extraction of the common drug compounds by the extracting agents with different pH values and the like, greatly overcomes the analysis errors caused by multiple measurements of different components, and improves the contrast and the method stability among different components of trace residual human and animal drug compounds in sediment samples. With the increase of available drug compound standard substances, the range of applicable drug compounds can be further expanded.)

1. A high-flux high-precision detection method for human and veterinary drug residues in sediments is characterized in that the flow is as follows: collecting and storing a sediment sample (1), pretreating the sample (2), extracting and enriching a solid phase (3), blowing nitrogen and concentrating (4), fixing the volume (5), measuring by a high performance liquid chromatography tandem mass spectrometer (6), analyzing data (7) and determining the application range of the method (8);

the method processes (1), (2), (3), (4), (5), (6), (7) and (8) are carried out in sequence, and deletion or bit sequence adjustment cannot be carried out.

2. The process flow (1) according to claim 1, characterized in that: fresh sediment samples were collected in glass bottles after baking at 450 ℃ and storage at-20 ℃ in the dark.

3. The process flow (2) according to claim 1, characterized in that: freeze drying and grinding (21), adding isotope internal standard (22), extracting (23), adjusting pH (24), adding disodium ethylene diamine tetraacetate dihydrate (25);

the method comprises the steps of (21) freeze-drying the frozen sediment sample to be dry, and grinding the sediment sample through a 0.15mm sieve;

in the method, 2g of sediment sample (accurate to 0.0001 g) is accurately weighed, and the amount of each added isotope internal standard corresponding to the human and animal medicine components is 100 ng;

in the process flow (23), the extracting agents are phosphate buffer solution and acetonitrile; the pH of the phosphate buffer solution is 3.0 and 9.0 respectively in the two methods; the extraction step comprises three steps of ultrasonic extraction, namely 10mL of phosphate buffer solution and 20mL of acetonitrile in the first step, 6mL of phosphate buffer solution and 12mL of acetonitrile in the second step, and 4mL of phosphate buffer solution and 8mL of acetonitrile in the third step; uniformly mixing the extraction liquid extracted in the three steps, and metering the volume to 800mL by using ultrapure water;

the pH of the extract liquid with constant volume in the process flow (24) is respectively adjusted to 3.0 and 9.0;

in the method, the volume of the extract liquid with constant volume in the flow (25) is 1g, and the amount of the disodium ethylene diamine tetraacetate dihydrate is added.

4. The process flow (3) according to claim 1, characterized in that: activating a hydrophilic lipophilic balance solid phase extraction column (specification is 500mg, 6 mL) (31);

the process scheme (31) was activated with 10mL of chromatographically pure methanol followed by 10mL of ultrapure water.

5. The process (4) according to claim 1, characterized in that it comprises: leaching (41), drying water (42) by nitrogen blowing, and eluting (43);

in the method flow (41), 10mL of ultrapure water is used for leaching the solid-phase extraction column;

in the method, the water in the solid phase extraction column is dried by nitrogen blowing by a water bath nitrogen blowing instrument;

the process scheme (43) uses 12mL of chromatographically pure methanol to elute the solid phase extraction column.

6. The process flow (5) according to claim 1, characterized in that: the volume was reduced to 1mL using chromatographically pure methanol-water (1: 1, vol.).

7. The process flow (6) according to claim 1, characterized in that: performing liquid chromatography separation by adopting a gradient elution method, wherein a mobile phase is an organic phase: chromatographically pure methanol; water phase: 0.1% aqueous formic acid + 2mmol/L ammonium acetate solution, flow rate: and (3) 0.3mL/min, and performing scanning monitoring in a positive ion mode and a negative ion mode by tandem mass spectrometry at the same time.

8. The process flow (7) according to claim 1, characterized in that: quantifying by adopting an internal standard method; the results of the extraction of the co-drug compounds by the extractants of different pH can be cross-checked.

9. The process flow (8) according to claim 1, characterized in that: the method is suitable for detecting 6 kinds of 86 kinds of human and animal medicine compounds in the sediment, and the range of the applicable medicine compounds can be further expanded along with the increase of the available medicine compound standard substances.

Technical Field

The invention belongs to the technical field of environmental monitoring, and particularly relates to a high-flux high-precision detection method for human and animal drug residues in sediments.

Background

In recent years, human and animal medicines are widely concerned as emerging pollutants due to the characteristics of continuous and large-scale use, increased environmental exposure, potential harm to water environment and human health and the like. The medicaments are frequently used in large quantities for human and veterinary drug therapy, as well as growth promoters in the processes of beekeeping, livestock and aquaculture, and the like. Due to the great difference of the drug intake efficiency of different organisms and the limited drug treatment technology in the sewage treatment plant, a great amount of drug residues directly or indirectly enter the water environment through the discharge modes of effluent water of the sewage treatment plant, domestic sewage, livestock and poultry and aquaculture wastewater and the like. Eventually, the continuous exposure of these drugs to water environments can have serious negative effects on the ecosystem and human health, such as resistance gene stress, endocrine disruption, and alteration of microbial community architecture.

Because human and animal drugs exist in trace or even ultra-trace levels in an environmental medium, the variety of compounds is various, the physicochemical properties are different, and the matrix of an environmental sample is complex, which brings challenges to human and animal drug detection. The United states environmental protection agency provides a standard analysis program (EPA 1694) for drugs and personal care products (PPCPs), which is used for reference by numerous countries, but the high-precision trace/trace high-throughput analysis technology for human and animal drug residues in environmental media needs to be improved, China is still at the reference and applicability improvement level at present, a standard method technical system and an environmental quality standard are not formed, and the discrimination, prevention and control and management of the drug residue environmental pollution problem are restricted. In conclusion, the patent provides a high-throughput high-precision detection method for trace residues of 6 kinds of 86 human and animal medicaments in sediments, and a specific medicament list is shown in figure 1.

Disclosure of Invention

The invention provides a high-flux high-precision detection method for human and animal drug residues in sediment by optimizing an instrument analysis method and a pretreatment method aiming at the technical defects of complex pretreatment, low recovery rate, high detection limit and the like of high-flux trace detection of the human and animal drug residues in the sediment. With the increase of available drug compound standard substances, the range of applicable drug compounds can be further expanded.

The invention provides a high-flux high-precision detection method for human and animal drug residues in sediments, which comprises the following steps: the method comprises the steps of (1) collecting and storing sediment samples, (2) preprocessing the samples, (3) solid phase extraction and enrichment, (4) nitrogen blowing and concentration, (5) constant volume, (6) high performance liquid chromatography tandem mass spectrometry (HPLC-MS), data analysis (7) and method application range (8). The method is sequentially carried out, and deletion or bit sequence adjustment cannot be carried out.

The invention solves the key technical problems of precision improvement and multi-component high-efficiency measurement by the following scheme:

the isotope internal standard corresponding to the human and animal medicine components is added in the process flow (2) of the method, so that the loss of the medicine components in the experimental process can be eliminated;

the method has the advantages that the process (2) selects proper extracting agents (phosphate buffer solution and acetonitrile) and the extracting step improves the extracting efficiency of the medicine components with different physicochemical properties in the sediment;

the method comprises the following process (2) adding disodium ethylene diamine tetraacetate dihydrate to complex Ca²⁺And Mg²⁺Alkali metal ions and trace heavy metal ions are obtained, so that the extraction efficiency of macrolide and tetracycline antibiotics is improved;

the method has the advantages that the flow (6) selects proper mobile phase composition and gradient elution procedures to improve the separation efficiency of the human and animal medicines;

in the method, the detection results of the extraction of the common drug compound by the extractants with different pH values in the process (7) are cross-checked, so that the stability of the method and the contrast of the detection results can be improved;

the method of the invention is now suitable for detecting 6 kinds of 86 kinds of human and animal medicine compounds in sediments, and can further expand the range of the applicable medicine compounds along with the increase of the available standard substances of the medicine compounds.

Drawings

Figure 1 is a list of 6 broad categories of 86 human and veterinary drugs.

FIG. 2 is a flow chart of the main steps of the method of the present invention.

Figure 3 total ion flowsheet of human veterinary drug and its isotopic internal standard in positive ion mode (a) and negative ion mode (B).

Detailed Description

The specific implementation mode of the high-throughput high-precision detection method for the multi-component residue of the human and animal medicines in the sediment is shown in figure 2, and the flow of the method comprises the following steps: the method comprises the steps of (1) collecting and storing sediment samples, (2) preprocessing the samples, (3) solid phase extraction and enrichment, (4) nitrogen blowing and concentration, (5) constant volume, (6) high performance liquid chromatography tandem mass spectrometry (HPLC-MS), data analysis (7) and method application range (8). The method is sequentially carried out, and deletion or bit sequence adjustment cannot be carried out.

Sediment sample collection and preservation (1): samples of the sediment were collected in glass bottles after baking at 450 ℃ and stored at-20 ℃ in the dark.

Sample pretreatment (2): freeze drying the sediment sample, grinding the sediment sample through a 0.15mm sieve; accurately weighing 2g of sediment sample (accurate to 0.0001 g) in a 35mL centrifuge tube, adding 100ng of isotope internal standard into the centrifuge tube, and standing overnight at 4 ℃; performing ultrasonic extraction in three steps by using phosphate buffer solution and acetonitrile, mixing three extraction solutions, performing constant volume to 800mL by using ultrapure water, adjusting pH, adding Na₂EDTA·2H₂O, and mixing well.

Solid phase extraction enrichment (3): the HLB solid phase extraction column (500 mg, 6 mL) was activated with 10mL of chromatographically pure methanol and 10mL of ultrapure water in this order, and the sample solution was transferred to a small column, and after the transfer, the sample bottle was rinsed with 2X 50mL of analytically pure methanol-water (5: 95, vol.).

Nitrogen-blown concentration (4): leaching the small column by using 2X 5mL of ultrapure water, and discarding the filtrate; nitrogen blowing is carried out on the water in the HLB solid-phase extraction column by using a water bath nitrogen blowing instrument; elution with 12mL of chromatographically pure methanol; the eluent was evaporated to dryness in a 35 ℃ water bath using a water bath nitrogen blower and high purity nitrogen.

Volume (5): using chromatographically pure methanol-water (1: 1, volume ratio) to fix the volume to 1mL, filtering the mixture by a 0.2 mu m needle filter to a chromatographic bottle, and waiting for the determination of a high performance liquid chromatography tandem mass spectrometer.

High performance liquid chromatography tandem mass spectrometry (6):

1. high performance liquid chromatography conditions

a) A chromatographic column: kinetex C18, 100 mm. times.2.1 mm. times.2.6 μm;

b) mobile phase: organic phase: chromatographically pure methanol; water phase: 0.1% formic acid water + 2mmol/L ammonium acetate solution;

c) flow rate: 0.3 mL/min; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution;

d) gradient elution procedure: 0-1.0min, the organic phase ratio is kept 15%; 1.0-2.0min, the proportion of the organic phase is increased linearly from 15 percent to 30 percent; 2.0-5.0min, the organic phase proportion linearly increases from 30% to 40%; 5.0-10.0min, the organic phase ratio linearly increased from 40% to 50%; 10.0-14.0min, the organic phase proportion linearly increases from 50% to 70%; 14.0-16.0min, the proportion of the organic phase is linearly increased from 70% to 100%; 16.0-20.0min, the organic phase proportion is kept 100%; 20.0-25.0min, the organic phase proportion is kept 15%; ending 25.0 min;

2. tandem mass spectrometry conditions

a) An analytical instrument: agilent high performance liquid chromatography-tandem mass spectrometry (ABI 6500Q TRAP HPLC/MS/MSSystem);

b) an ion source: electrospray ionization source (ESI);

c) the scanning mode is as follows: multiple reaction monitoring mode (MRM), positive ion mode and negative ion mode scanning monitoring simultaneously;

d) air curtain air: 25 psi; collision gas: 9 psi; atomizing: 50 psi; drying gas: 60 psi;

e) ion source spray voltage: 5500V (positive ion mode), -4500V (negative ion mode);

f) mass spectrometry ion source temperature: 650 ℃;

h) the gas curtain gas and the collision gas are high-purity nitrogen; the atomization gas is zero-order air; the drying gas is clean dry air.

Data analysis (7): quantifying by adopting an internal standard method; the results of the extraction of the co-drug compounds by the extractants of different pH can be cross-checked.

Method application range (8): the method is suitable for detecting 6 kinds of 86 human and animal medicine compounds in the sediment, and the range of the applicable medicine compounds can be further expanded along with the increase of the available standard substances of the medicine compounds; description of the method accuracy: with reference to the method requirements of GB/T6379.1 and GB/T6379.2, the surface water method (pH 3 + 1 gNa) is established₂EDTA·2H₂O) the average recovery rate is 50-150% within the range of 10ng/g addition level, and the relative standard deviation is less than 19%; the average recovery rate in the range of 100ng/g addition level is 50-150%, and the relative standard deviation is less than 17%. Established surface Water method (pH 9 + 1g Na)₂EDTA·2H₂O) the average recovery rate is 50-149% in the range of 10ng/g addition level, and the relative standard deviation is less than 28%; the average recovery rate in the range of 100ng/g addition level is 53-149%, and the relative standard deviation is less than 18%.