Pretreatment method universally applicable to detection of perfluorinated and polyfluoroalkyl substances in biological sample

Abstract

The invention relates to the technical field of chemical detection, in particular to a pretreatment method universally applicable to detection of perfluorinated and polyfluoroalkyl substances in biological samples. Comprising the following steps: (1) adding an isotope-labeled internal standard solution and acetonitrile into a biological sample, carrying out vortex mixing, and centrifugally extracting supernate; (2) carrying out nitrogen blowing on the supernate to remove acetonitrile, adding pure water to a constant volume, and then carrying out solid-phase extraction through an activated WAX solid-phase extraction column; and (3) carrying out elution treatment on the WAX solid-phase extraction column, collecting an eluent, carrying out nitrogen blowing until the eluent is nearly dry, and redissolving the eluent with a methanol aqueous solution to obtain a sample to be detected. According to the present invention, the protein removal is performed on the sample through the acetonitrile precipitation protein property, the enrichment concentration is performed on the PFASs in the biological sample through the solid phase extraction technology, and the quantification limit and the detection limit of the subsequent instrument detection are reduced; the method is easy to operate, low in cost, short in consumed time and easy to implement and popularize.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical detection, and in particular to a pretreatment method generally applicable to the detection of per- and polyfluoroalkyl substances in biological samples. Background Art

[0002] Per- and polyfluoroalkyl substances (PFASs) are widely used in industrial production and daily life due to their unique chemical structure and excellent properties, such as in textiles, firefighting foams, and food packaging. However, PFASs are persistent, bioaccumulative, and toxic, making them difficult to degrade in the environment and entering the human body through the food chain. Once in the human body, they can cause cardiovascular toxicity, hepatotoxicity, metabolic toxicity, carcinogenicity, endocrine and immune disruption, and reproductive toxicity. They can not only directly affect reproduction and development, liver and kidney function, and the immune system, but also have the potential to be carcinogenic and promote cancer development.

[0003] PFASs are widely present in biological samples, and instrumental analysis is easily interfered with by fatty compounds, proteins, or other organic compounds. It is usually necessary to select appropriate extraction and purification methods for sample pretreatment. Currently, liquid-liquid extraction, solid-phase extraction, and gel permeation chromatography (GPC) purification are more common. Liquid-liquid extraction has a limited enrichment factor, resulting in a high instrument detection limit and difficulty in detecting pollutants, and is currently less used. Gel permeation chromatography purification, on the other hand, has a relatively small scope of application due to its complex operating steps and high cost.

[0004] Compared to other methods, solid-phase extraction (SPE) offers high enrichment multiples, is relatively simple to operate, and is widely used. Duan et al. used β-glucuronidase / carboxylase to hydrolyze proteins before SPE, but this method is time-consuming, costly, and produces poor removal results.

[0005] Therefore, there is an urgent need to develop an efficient pretreatment method for PFASs in biological samples. This method should have the characteristics of high purification efficiency, simple operation, high enrichment multiple and low cost to meet the needs of accurate detection of PFASs in biological samples. Summary of the Invention

[0006] The present invention aims to provide a pretreatment method generally applicable to the detection of per- and polyfluoroalkyl substances in biological samples for analysis by ultra-high performance liquid chromatography-tandem mass spectrometry. This pretreatment method is suitable for quantification using either internal or external standard methods. This method removes protein from the sample through acetonitrile precipitation and enriches and concentrates PFASs in the biological sample using solid-phase extraction, thereby reducing the limits of quantification and detection for subsequent instrumental analysis. This method is simple to operate, inexpensive, and time-consuming, making it easy to implement and promote.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a pretreatment method generally applicable to the detection of per- and polyfluoroalkyl substances in biological samples, the method comprising the following steps:

[0009] (1) Add acetonitrile to the biological sample, vortex mix, and then centrifuge to extract the supernatant;

[0010] (2) The supernatant was nitrogen-purged to remove acetonitrile, and pure water was added to the volume, followed by solid-phase extraction through an activated WAX solid-phase extraction column;

[0011] (3) The WAX ​​solid phase extraction column was eluted, the eluate was collected, blown to near dryness with nitrogen, and redissolved with methanol-water solution to obtain the sample to be tested.

[0012] In the above technical solution, further, in step (1), the volume ratio of the biological sample to acetonitrile is 1:4.

[0013] In the above technical solution, further, in step (1), the vortex time is 2 minutes; the centrifugal speed is 5000-6000 rpm, and the time is 20-30 minutes.

[0014] In the above technical solution, further, in step (2), the activation treatment is: activating the WAX ​​solid phase extraction column with methanol containing 0.28 vol% ammonia water, methanol and pure water in sequence.

[0015] In the above technical solution, further, in step (3), the elution treatment is: eluting the WAX ​​solid phase extraction column with an acetic acid-ammonium acetate buffer solution with a pH of 4.5 and pure water, discarding the effluent, and then eluting with methanol and methanol containing 0.28 vol% ammonia water in sequence, and collecting the eluate.

[0016] In the above technical solution, further, in step (3), the acetic acid-ammonium acetate buffer solution is a mixed solution of acetic acid and ammonium acetate, and the concentration of the ammonium acetate is 25 mM.

[0017] In the above technical solution, further, in step (3), the concentration of the methanol aqueous solution is 50 vol%.

[0018] The beneficial effects of the present invention are:

[0019] 1) The pretreatment method provided by the present invention has a better recovery rate for PFASs of carboxylic acid sulfonic acid. Compared with other methods in the prior art, it has the advantages of relatively low cost, short time consumption, simple operation and good experimental treatment effect;

[0020] 2) The present invention removes protein from the sample by acetonitrile precipitation, which is less expensive and has a better recovery rate than the enzymatic protein hydrolysis method in the prior art;

[0021] 3) The present invention uses solid-phase extraction technology to enrich and concentrate PFASs in biological samples, effectively reducing the quantitative limit and detection limit of subsequent instrument detection, and solving the problem of insufficient detection sensitivity caused by the limited enrichment multiple of liquid-liquid extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the recovery rate of PFASs in Example 1;

[0023] Figure 2 This is the recovery rate of PFASs in Comparative Example 1. DETAILED DESCRIPTION

[0024] The following examples are provided to help those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. All reagents and materials used, unless otherwise specified, can be obtained from commercial sources.

[0025] Example 1

[0026] The pretreatment method in this example was applied to the calf serum sample, and the spiked recoveries of the following 16 PFASs were quantitatively determined using the internal standard method.

[0027] The internal standard compounds corresponding to the target substances in this example are shown in Table 1.

[0028] Table 1 Internal standard compounds corresponding to target substances

[0029]

[0030]

[0031] Sample blank: Take 0.5 ml of calf serum, add 2 ml of acetonitrile thereto, vortex mix for 2 min, centrifuge at 6000 rpm for 30 min, place the supernatant in a new PP tube and concentrate to 0.5 ml under nitrogen at 30 ° C. Solid phase extraction is performed with CNWBOND WAX SPE (500 mg, 6 ml), and the column is activated with 6 ml of methanol containing 0.28 vol% ammonia, 6 ml of methanol and 6 ml of pure water in sequence. The sample is loaded, and after loading, the column is washed with 6 ml of 25 mM acetic acid-ammonium acetate buffer solution with pH 4.5 and 10 ml of pure water in sequence. The effluent is discarded, and the column is eluted with 15 ml of methanol and 15 ml of methanol containing 0.28 vol% ammonia in sequence. The eluate is collected and concentrated to dryness under nitrogen at 30 ° C. 2.5 ng (50 μL) is added. 50ppb) internal standard and 450 μL methanol-water solution (1:1, v / v) were reconstituted, mixed and the solution was transferred into the injection bottle for injection analysis.

[0032] Sample plus standard: Take 0.5 ml of calf serum, add 5 ng of PFASs standard and 2 ml of acetonitrile, vortex mix for 2 minutes, centrifuge at 6000 rpm for 30 minutes, place the supernatant in a new PP tube and concentrate to 0.5 ml under nitrogen at 30 ° C. Solid phase extraction is performed with CNWBOND WAX SPE (500 mg, 6 ml). The column is activated with 6 ml of methanol containing 0.28 vol% ammonia, 6 ml of methanol and 6 ml of pure water in sequence. The sample is loaded. After loading, the column is washed with 6 ml of 25 mM acetic acid-ammonium acetate buffer solution with pH 4.5 and 10 ml of pure water in sequence. The effluent is discarded, and the column is eluted with 15 ml of methanol and 15 ml of methanol containing 0.28 vol% ammonia in sequence. The eluate is collected and concentrated to dryness under nitrogen at 30 ° C. 2.5 ng (50 μL) is added. 50 ppb) internal standard and 450 μL methanol-water solution (1:1, v / v) were reconstituted, mixed and transferred into a sampling vial for injection analysis.

[0033] 10 ppb concentration standard: Take 50 μL of the standard stock solution (2000 ppb) and add 950 μL of methanol to prepare a 100 ppb standard. Take 50 μL of the 100 ppb standard and add 2.5 ng (50 μL of 50 ppb) internal standard and 400 μL of methanol to prepare 0.5 mL of 10 ppb standard containing 5 ppb internal standard for injection and analysis.

[0034] Samples were separated using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) on a Hypersil GOLD column (Thermo Fisher Scientific, 1.5 μm, 2.1 x 100 mm). Mobile phases A (aqueous) and B (methanolic) both contained 2 mmol / L ammonium acetate and 2 mmol / L N-methylpiperidine. The column temperature was set at 40°C, and the injection plate temperature was set at 8°C. The elution schedule is shown in Table 2.

[0035] Table 2 Elution program

[0036] Time (min) Flow rate (mL / min) %B (methanol containing additives) %A(water containing additives) 0.000 0.300 30.0 70.0 0.500 0.300 30.0 70.0 1.500 0.300 50.0 50.0 6.500 0.300 60.0 40.0 20.500 0.300 75.0 25.0 23.500 0.300 85.0 15.0 24.000 0.300 100.0 100.0 29.000 0.300 100.0 100.0 29.100 0.300 30.0 70.0 35.600 0.300 30.0 70.0

[0037] Mass spectrometry was performed in the negative ion mode (ESI - ), full scan (Full MS) and data dependent mass spectrometry (dd-MS 2 ) in combination with a scanning range of m / z 100-1000.

[0038] The recovery rate was calculated according to the above process: Recovery rate (%) = (measured concentration of spiked sample - measured concentration of blank sample) / measured concentration of standard product × 100%.

[0039] like Figure 1 As shown, the recovery rate of carboxylic acid sulfonic acid PFASs is relatively good, with most recoveries exceeding 80%. Short-chain PFASs are more water-soluble than long-chain PFASs, resulting in lower retention rates on solid-phase extraction columns and lower recoveries.

[0040] Comparative Example 1

[0041] Sample preparation was performed using the method published by Duan et al. to hydrolyze proteins with β-glucuronidase / carboxylase before solid-phase extraction (https: / / doi.org / 10.1016 / j.envint.2019.105295).

[0042] like Figure 2 Compared with the method of the present invention, the method of using enzyme to hydrolyze protein is time-consuming, expensive and has poor experimental results.

[0043] In summary, the present invention uses solid-phase extraction to treat PFASs in biological samples, offering advantages such as relatively low cost, short time consumption, simple operation, and excellent experimental results. This provides a more accurate and convenient pretreatment method for PFAS detection, and a powerful tool for subsequent PFAS detection in biological samples.

[0044] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. The scope of protection of the present invention shall be determined by the scope defined in the claims. Other variations or modifications may be made based on the above description. Obvious variations or modifications derived therefrom shall remain within the scope of protection of the present invention.

Claims

1. A pretreatment method generally applicable to the detection of per- and polyfluoroalkyl substances in biological samples, characterized in that: The method comprises the following steps: (1) Add acetonitrile to the biological sample, vortex mix, and then centrifuge to extract the supernatant; (2) The supernatant was nitrogen-purged to remove acetonitrile, and pure water was added to the volume, followed by solid-phase extraction through an activated WAX solid-phase extraction column; (3) The WAX ​​solid phase extraction column was eluted, the eluate was collected, blown to near dryness with nitrogen, and redissolved with methanol-water solution to obtain the sample to be tested.

2. The pre-treatment method according to claim 1, characterized in that In step (1), the volume ratio of the biological sample to acetonitrile is 1:

4.

3. The pre-treatment method according to claim 1, characterized in that In step (1), the vortex time is 2 minutes; The centrifugal speed is 5000-6000 rpm, and the time is 20-30 min.

4. The pre-treatment method according to claim 1, characterized in that: In step (2), the activation treatment is: sequentially activating the WAX ​​solid phase extraction column with methanol containing 0.28 vol% ammonia water, methanol and pure water.

5. The pre-treatment method according to claim 1, characterized in that: In step (3), the elution treatment is: eluting the WAX ​​solid phase extraction column with an acetic acid-ammonium acetate buffer solution with a pH of 4.5 and pure water, discarding the effluent, and then eluting with methanol and methanol containing 0.28 vol% ammonia water in sequence, and collecting the eluate.

6. The pre-treatment method according to claim 5, characterized in that: In step (3), the acetic acid-ammonium acetate buffer solution is a mixed solution of acetic acid and ammonium acetate, and the concentration of the ammonium acetate is 25 mM.

7. The pre-treatment method according to claim 1, characterized in that: In step (3), the concentration of the methanol aqueous solution is 50 vol%.

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