A rapid sample pretreatment method for detecting environmental pollutants in biological samples

Abstract

This invention discloses a rapid sample pretreatment method for detecting environmental pollutants in biological samples, comprising the following steps: transferring the biological sample to be tested into a glass centrifuge tube, diluting it with ultrapure water, then sequentially adding an extraction internal standard, formic acid, and acetonitrile, mixing with a vortex mixer, adding an organic solvent, and continuing to extract the mixture with a vortex mixer to obtain a mixed sample; centrifuging the centrifuge tube containing the mixed sample, then transferring the upper organic phase, repeating the operation three times, combining the organic phases and concentrating them by purging with nitrogen; purging the concentrated sample using a solid-phase extraction column to elute the target pollutant, collecting the eluent, purging it to near dryness with nitrogen, adding the injection internal standard, and then analyzing the sample using gas chromatography-mass spectrometry.

Description

Technical Field

[0001] This invention relates to the field of bioanalytical chemistry, and more specifically, to a rapid sample pretreatment method for detecting environmental pollutants in biological samples. Background Technology

[0002] Humans are exposed to complex mixtures of chemicals through various pathways, including air, water, and the food chain. These substances include polycyclic aromatic hydrocarbons (PAHs), dioxins, pesticides, organophosphates, polychlorinated biphenyls (PCBs), and perfluorinated and polyfluorinated compounds (PFOCs). Studies have shown that these chemicals are closely linked to various cancers and other health problems in humans and laboratory animals. However, due to limitations in current analytical techniques, research data on the cumulative exposure effects of these pollutants and their toxicological interactions remain scarce.

[0003] Environmental pollutants in biological samples are typically present at extremely low concentrations (e.g., ng / mL or pg / mL), making direct detection often difficult to achieve the sensitivity required by the instruments. Furthermore, biological samples contain a large number of complex matrix components, such as proteins, lipids, salts, and metabolites, which can interfere with the detection of target pollutants, leading to inaccurate analytical results. Therefore, in toxicological analysis, the pretreatment process needs to remove or reduce these interfering substances to improve the purity and detection sensitivity of the target pollutant. Concentration and enrichment are also necessary to ensure that the concentration of the target pollutant reaches above the instrument's detection limit. Most methods developed in the past for determining environmental pollutants in biological samples employ protein precipitation (PP), liquid-liquid extraction (LLE), and solid-phase extraction (SPE). While PP and LLE are simple to operate, insufficient sample purification can lead to instrument contamination and unreliable quantitative results. Although SPE offers high selectivity and may contain fewer interfering substances in the extract than PP or LLE, its operation is more cumbersome and time-consuming. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned deficiencies of existing technologies and provide a simple, rapid, sensitive, and reproducible sample pretreatment method for the detection of environmental pollutants in biological samples. This method is used to extract more than 700 environmental pollutants of high toxicological concern from biological samples, and its practical application value has been verified by GC-Orbitrap MS. This method has sufficient sensitivity and can provide reproducible quantitative analysis for all analytes studied over a wide concentration range.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A rapid sample pretreatment method for detecting environmental pollutants in biological samples includes the following steps:

[0007] (1) Transfer the biological sample to be tested to a glass centrifuge tube, dilute it with ultrapure water, add the extraction internal standard, formic acid and acetonitrile in sequence, mix with a vortex mixer, add organic solvent, and continue to extract the mixture with a vortex mixer to obtain the mixture sample;

[0008] (2) Centrifuge the centrifuge tube containing the mixture sample, then remove the upper organic phase, repeat the operation three times, combine the organic phases and concentrate them by purging with nitrogen.

[0009] (3) The concentrated sample was purified by using a solid phase extraction column to elute the target pollutant. The eluent was collected and purged with nitrogen until nearly dry. An internal standard was added, and the sample was then analyzed by gas chromatography-mass spectrometry.

[0010] Optionally, in step (1), the biological sample to be tested includes at least one of cell lysate, serum, whole blood, and tissue homogenate.

[0011] Optionally, in step (1), the volume ratio of the biological sample to be tested to the ultrapure water is 1:0.5 to 1:1; the mixing time is 10 min to 1 h; and the extraction time is 0.5 h to 1 h.

[0012] Optionally, in step (1), the volume ratio of formic acid to acetonitrile in the mixture of formic acid and acetonitrile is 1:99 to 5:95.

[0013] Optionally, in step (1), the organic solvent used for extraction includes at least one of n-hexane, dichloromethane, petroleum ether, and methylcyclohexane.

[0014] Optionally, in step (1), the volume ratio of the biological sample to be tested and the mixture of formic acid and acetonitrile is 1:2 to 1:5; the amount of organic solvent added is 1 to 10 times the volume of the biological sample.

[0015] Optionally, in step (2), the centrifugation speed is 3500g / min to 4000g / min, the time is 5min to 10min, and the temperature is 4℃.

[0016] Optionally, in step (3), the solid phase extraction column is at least one of C18, HLB and Florisil columns.

[0017] Optionally, in step (3), the elution solution used is a mixed solution of n-hexane and dichloromethane, wherein the volume ratio of n-hexane to dichloromethane in the mixed solution is 1:1 to 1:4, and the elution volume is 10 mL to 30 mL.

[0018] Implementing the embodiments of the present invention will have the following beneficial effects:

[0019] (1) This invention requires a small sample volume, is simple and easy to operate, and has a fast and efficient extraction speed. The separated organic solvent is effectively purified and impurities are removed. This pretreatment method is applicable to various biological samples, which greatly facilitates subsequent toxicity studies.

[0020] (2) This invention employs organic solvent extraction, a method that is highly flexible and easy to implement. Typically, simply adding an organic solvent to the sample and vortexing it thoroughly is sufficient to obtain good extraction of the target compound. Simultaneously, the stable recovery rate of the isotope internal standard indicates that the subsequent purification process does not affect the content of environmental pollutants, ensuring the accuracy of the detection results. The pretreatment results of this invention have good effects and high reliability and stability. Attached Figure Description

[0021] Figure 1 The recovery results of isotope internal standards in biological samples (EI source).

[0022] Figure 2 The recovery results of isotope internal standards in biological samples (CI source).

[0023] Figure 3 This is the result of GC-MS detection of environmental pollutants. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0025] Example 1

[0026] The rapid sample pretreatment method for detecting environmental pollutants in biological samples in this embodiment includes the following steps:

[0027] (1) Sample preparation: Take the frozen serum out of the freezer and thaw it at 4°C until it returns to room temperature. Use a pipette to accurately measure 0.8 mL of serum and transfer it to a pre-cleaned 30 mL glass centrifuge tube. Add 0.8 mL of Millipore water (ultrapure water) to dilute it.

[0028] (2) Preparation of a 1% formic acid acetonitrile (v / v) solution: Accurately measure 99 mL of acetonitrile (chromatographic grade) using a graduated cylinder and pour it into a clean beaker. Accurately measure 1 mL of formic acid (chromatographic grade, purity ≥98%) using a pipette and slowly add it to the acetonitrile. Gently stir with a glass rod to ensure that the formic acid and acetonitrile are thoroughly mixed. Transfer the prepared solution to a brown bottle, seal it, and label the solution components (1% formic acid acetonitrile) and preparation date. Store it away from light.

[0029] (3) Sample extraction: Add 10 μL of [unspecified ingredient] to a 30 mL glass centrifuge tube. 13 A 100 ng / mL PCB isotope internal standard (68C-LCS) was used as an internal standard for subsequent quantitative analysis. 2 mL of 1% formic acid / acetonitrile (v / v) was added, and the mixture was extracted using a vortex mixer for 1 h. Then, 5 mL of n-hexane was added, and the mixture was extracted again using a vortex mixer for 1 h to ensure complete transfer of the target pollutant to the organic phase.

[0030] (4) Component collection: Centrifuge tubes containing the samples were placed in a centrifuge for separation at a speed of 4000 g / min for 5 min, with the centrifugation temperature strictly controlled at 4℃. The upper organic phase (n-hexane phase) was carefully transferred to a pre-cleaned glass centrifuge tube using a pipette. This extraction process was repeated three times, and the n-hexane phases were combined into the same glass centrifuge tube.

[0031] (5) Nitrogen blowing concentration: After extraction, the combined n-hexane extracts were placed in a nitrogen blower. The n-hexane extracts were evaporated to about 0.5 ml under a gentle nitrogen flow at 30 °C.

[0032] (6) Sample purification: The concentrated n-hexane sample was purified using a Florisil column (a commercial solid-phase extraction column). The target contaminant was eluted using n-hexane:dichloromethane (20 mL, 1:1, v / v) as the mobile phase. The eluent was collected and evaporated to near dryness under a gentle nitrogen stream at 30°C.

[0033] (7) GC-MS test: After evaporation to near dryness under nitrogen flow, 5 μL of [a specific substance] was added. 13 C PCB (68C-IS, 100 ng / mL) was stored in a 4°C refrigerator and awaited analysis by gas chromatography-mass spectrometry (GC-MS) electron impact ionization source (EI).

[0034] Example 2

[0035] The rapid sample pretreatment method for detecting environmental pollutants in biological samples in this embodiment includes the following steps:

[0036] (1) Sample preparation: Use a pipette to transfer 1 ml of tissue homogenate into a pre-cleaned 30 mL glass centrifuge tube and add 0.5 mL of Millipore water (ultrapure water) for dilution.

[0037] (2) Preparation of a 4% formic acid acetonitrile (v / v) solution: Accurately measure 96 mL of acetonitrile (chromatographic grade) using a graduated cylinder and pour it into a clean beaker. Accurately measure 4 mL of formic acid (chromatographic grade, purity ≥98%) using a pipette and slowly add it to the acetonitrile. Gently stir with a glass rod to ensure that the formic acid and acetonitrile are thoroughly mixed. Transfer the prepared solution to a brown bottle, seal it, and label the solution components (4% formic acid acetonitrile) and preparation date. Store it away from light.

[0038] (3) Sample extraction: Add 10 μL of [unspecified ingredient] to a 30 mL glass centrifuge tube. 13 C-transCD (100 ng / mL) internal standard extract was used as an internal standard for subsequent quantitative analysis. 4 mL of 4% formic acid / acetonitrile (v / v) was added for protein precipitation in serum. The mixture was extracted using a vortex mixer for 1 h, followed by the addition of 8 mL of n-hexane, and extraction was continued using a vortex mixer for another 1 h to ensure complete transfer of the target contaminant to the organic phase.

[0039] (4) Component collection: Centrifuge tubes containing the samples were placed in a centrifuge for separation at a speed of 4000 g / min for 10 min, with the centrifugation temperature strictly controlled at 4℃. The upper organic phase (n-hexane phase) was carefully transferred to a pre-cleaned glass centrifuge tube using a pipette. This extraction process was repeated three times, and the n-hexane phases were combined into the same glass centrifuge tube.

[0040] (5) Nitrogen blowing concentration: After extraction, the combined n-hexane extracts were placed in a nitrogen blower. The n-hexane extracts were evaporated to about 0.5 ml under a gentle nitrogen flow at 30 °C.

[0041] (6) Sample purification: The concentrated n-hexane sample was purified using a Florisil column (a commercial solid-phase extraction column). The target contaminant was eluted using a hexane:dichloromethane (30 mL, 1:1, v / v) mobile phase. The eluent was collected and evaporated to near dryness under a gentle nitrogen stream at 30 °C.

[0042] (7) GC-MS test: After evaporation to near dryness under nitrogen flow, 5 μL of [a specific substance] was added. 13 C-HCB (100 ng / mL) was stored in a 4°C refrigerator and awaited analysis by chemical ionization source (CI) of gas chromatography-mass spectrometry (GC-MS).

[0043] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A rapid sample pretreatment method for detecting environmental pollutants in biological samples, characterized in that, Includes the following steps: (1) Transfer the biological sample to be tested to a glass centrifuge tube, dilute it with ultrapure water, add the extraction internal standard, formic acid and acetonitrile in sequence, mix with a vortex mixer, add organic solvent, and continue to extract the mixture with a vortex mixer to obtain the mixture sample; (2) Centrifuge the centrifuge tube containing the mixture sample, then remove the upper organic phase, repeat the operation three times, combine the organic phases and concentrate them by purging with nitrogen; (3) The concentrated sample was purified by using a solid phase extraction column to elute the target pollutant. The eluent was collected and purged with nitrogen until nearly dry. An internal standard was added, and the sample was then analyzed by gas chromatography-mass spectrometry.

2. The rapid sample pretreatment method for detecting environmental pollutants in biological samples according to claim 1, characterized in that, In step (1), the biological sample to be tested includes at least one of cell disruption fluid, serum, whole blood, and tissue homogenate.

3. The rapid sample pretreatment method for detecting environmental pollutants in biological samples according to claim 1, characterized in that, In step (1), the volume ratio of the biological sample to be tested to the ultrapure water is 1:0.5 to 1:1; the mixing time is 10 min to 1 h; and the extraction time is 0.5 h to 1 h.

4. The rapid sample pretreatment method for detecting environmental pollutants in biological samples according to claim 1, characterized in that, In step (1), the volume ratio of formic acid to acetonitrile in the mixture of formic acid and acetonitrile is 1:99 to 5:

95.

5. The rapid sample pretreatment method for detecting environmental pollutants in biological samples according to claim 1, characterized in that, In step (1), the volume ratio of the biological sample to be tested and the mixture of formic acid and acetonitrile is 1:2 to 1:5; the amount of organic solvent added is 1 to 10 times the volume of the biological sample.

6. The rapid sample pretreatment method for detecting environmental pollutants in biological samples according to claim 1, characterized in that, In step (1), the organic solvent used for extraction includes at least one of n-hexane, dichloromethane, petroleum ether, and methylcyclohexane.

7. The rapid sample pretreatment method for detecting environmental pollutants in biological samples according to claim 1, characterized in that, In step (2), the centrifugation speed is 3500g / min to 4000g / min, the time is 5min to 10min, and the temperature is 4℃.

8. The rapid sample pretreatment method for detecting environmental pollutants in biological samples according to claim 1, characterized in that, In step (3), the solid phase extraction column is at least one of C18, HLB and Florisil columns.

9. The rapid sample pretreatment method for detecting environmental pollutants in biological samples according to claim 1, characterized in that, In step (3), the elution solution used is a mixed solution of n-hexane and dichloromethane, wherein the volume ratio of n-hexane to dichloromethane in the mixed solution is 1:1 to 1:4, and the elution volume is 10 mL to 30 mL.

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