Pretreatment method suitable for perfluorinated and polyfluoroalkyl compounds and organophosphorus ester in biological sample

Abstract

A pretreatment method suitable for perfluorinated and polyfluoroalkyl compounds and organophosphorus esters in a biological sample comprises the following steps: adding acetonitrile into the biological sample, adding an internal standard, carrying out vortex, centrifuging, extracting a supernatant, adding acetonitrile again, carrying out vortex, centrifuging, extracting a supernatant again, combining the two supernatants, and carrying out freeze-drying to obtain the perfluorinated and polyfluoroalkyl compounds and organophosphorus esters. And carrying out solid-phase extraction, nitrogen concentration, methanol redissolution and sample introduction analysis on the merged supernate by adopting a phospholipid small column. According to the pretreatment method, firstly, acetonitrile is used for removing protein in a biological sample, then a phospholipid removal small column is used for further purifying fat substances in a solution, it is ensured that a target analyte is not interfered, nitrogen concentration and methanol redissolution are conducted, the purity and detection sensitivity of the sample are improved, the recovery rate of PFASs and OPEs reaches the ideal range, and the method is suitable for industrial production. And reliability and effectiveness are realized.

Description

Technical Field

[0001] This invention relates to the field of chemical detection technology, and more particularly to a pretreatment method applicable to perfluorinated and polyfluoroalkyl compounds and organophosphates in biological samples. Background Technology

[0002] Per- and polyfluoroalkyl substances (PFASs), including perfluoroalkyl and polyfluoroalkyl substances, are widely used in industrial production and consumer products due to their chemical stability, water and oil repellency, and other properties. Examples include fire-fighting foam, metal plating, semiconductor manufacturing, waterproof clothing, and food packaging. PFASs are persistent, bioaccumulative, and potentially toxic. They are difficult to degrade in the environment and can enter the human body through the food chain, causing serious harm such as reproductive toxicity, cardiovascular toxicity, endocrine disruption, and immune interference. They can directly affect reproduction and development, and even damage the immune system and cause cancer.

[0003] Organophosphates (OPEs) are a class of organic compounds containing carbon-phosphorus bonds. They are widely used as flame retardants and plasticizers in foams, plastics, textiles, hydraulic fluids, and various building materials. They are ubiquitous in the global environment and can be absorbed by organisms and humans through contact with indoor dust, inhalation, and the food chain, exhibiting various toxic effects such as endocrine disruption, neurotoxicity, and reproductive and developmental toxicity.

[0004] Existing pretreatment methods often use solid-phase extraction (SPE) columns to enrich PFASs and OPEs. However, since the SPE columns used for PFASs are mixed-type weak anion exchange columns, and those used for OPEs are mostly amino-based SPE columns, simultaneous detection of perfluorinated and polyfluoroalkyl substances and organophosphates is not possible. To address this, a pretreatment method suitable for perfluorinated and polyfluoroalkyl compounds and organophosphates in biological samples is proposed. This pretreatment method involves precipitating proteins in the biological sample, followed by separate extraction to enrich and concentrate the contaminants, providing a lower detection limit for subsequent substance detection. This method is highly efficient and easy to operate. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a pretreatment method universally applicable to the detection of various contaminants in biological samples, comprising sample purification and enrichment / concentration. Purification utilizes the property of acetonitrile to precipitate proteins, removing proteins from the sample. Nitrogen concentration is then used to enrich and concentrate the substances, lowering the limits of quantitation and detection for subsequent instrumental analysis. This method is simple to operate, inexpensive, saves samples, and is time-efficient, making it easy to implement and promote.

[0006] According to one aspect of the present invention, a pretreatment method for perfluorinated and polyfluoroalkyl compounds and organophosphates in biological samples is provided. The pretreatment method includes the following steps: adding acetonitrile and an internal standard to the biological sample, vortexing and centrifuging, extracting the supernatant, adding acetonitrile again, vortexing and centrifuging, extracting the supernatant again, combining the two supernatants, performing solid-phase extraction on the combined supernatant using a phospholipid column, concentrating with nitrogen, redissolving with methanol, and injecting for analysis.

[0007] Based on the above technical solution, the mass ratio of the biological sample to acetonitrile is 0.5~1:4.5~6.5, preferably 1:4.7.

[0008] Based on the above technical solution, the biological sample is a placental cryogenic sample.

[0009] Based on the above technical solution, the vortex duration is 1~2 minutes.

[0010] Based on the above technical solution, the centrifugation conditions are as follows: The centrifuge speed is 5000~6000 rpm; The centrifugation time is 10-20 minutes.

[0011] Based on the above technical solution, the phospholipid column is CNWBOND dephospholipid PPR Pro, 300 mg 3 mL.

[0012] Based on the above technical solution, the nitrogen concentration temperature is 30~40℃; The nitrogen concentration time is 3-4 hours.

[0013] Based on the above technical solution, the conditions for sample injection analysis are as follows: PFASs sample analysis is performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), with a Hypersil GOLD liquid chromatography column (Thermo Fisher Scientific, 1.5 μm, 2.1 x 100 mm) for sample separation. Mobile phase A (aqueous phase) and mobile phase B (methanol phase) both contain 2 mmol / L ammonium acetate and 2 mmol / L N-methylpiperidine. The PFASs elution process is as follows: the flow rate is 0.3 mL / min for both phases. 30%B, 70%A, 0~1.5 min; 50%B, 50%A, 1.5 min; 60%B, 40%A, 6.5 min; 75%B, 25%A, 20.5 min; 85%B, 15%A, 23.5 min; 100%B, 0%A, 24 min; 0%B, 100%A, 29 min; 30%B, 70%A, 29.1~35.6 min; The OPEs elution process is as follows: the flow rate is 0.4 mL / min; 30%B, 70%A, 0~1 min; 50%B, 50%A, 1 min; 100%B, 0%A, 9 min; 0%B, 100%A, 16.8 min; 30%B, 70%A, 17~20 min; The column temperature was set to 40℃, the injection plate temperature was set to 8℃, and the mass spectrometry analysis used electrospray ionization. PFASs were in negative ion mode (ESI-) and OPEs were in positive ion mode (ESI+). Full scan (Full MS) and data-dependent mass spectrometry (dd-MS2) were used together. The full scan range was 100-1000 m / z. Attached Figure Description

[0014] Figure 1 The detection results of PFASs in Embodiment 1 of the present invention; Figure 2 The results of OPE detection in Embodiment 1 of the present invention are shown.

[0015] Beneficial effects The technical solution of this invention solves the problems of high cost, long time consumption, and cumbersome operation in the existing methods for simultaneously extracting PFASs and OPEs, as well as the difficulty in obtaining samples and the inability to detect two substances in a single sample. To address this, this invention uses acetonitrile to remove proteins from placental samples, and then further refines the lipid substances in the sample through phospholipid column extraction to ensure that the target analytes are not interfered with. Nitrogen concentration and methanol redissolution are then performed to improve the purity of the sample and the detection sensitivity. Test results show that the technical solution adopted in this invention achieves the ideal recovery rate of both PFASs and OPEs, and the method is reliable and effective. Detailed Implementation

[0016] The following examples are intended to provide a more comprehensive understanding of the present invention by those skilled in the art, but do not limit the invention in any way. Unless otherwise specified, all reagents and materials used are commercially available. The placental samples without infectious diseases were obtained from Shanghai Fengxian Hospital. The placental samples were freeze-dried at -20 to -45°C for more than 24 hours, dried in a vacuum dryer until free of moisture, and then ground into powder using a mortar and pestle to obtain placental freeze-dried powder.

[0017] Example 1 The pretreatment method of this embodiment was used to detect placental lyophilized powder, and the spiked recovery rate of the following target substances was determined using the internal standard method.

[0018] The target substances and corresponding internal standard compounds in this embodiment are shown in Tables 1 and 2.

[0019] Table 1 lists the internal standard compounds corresponding to the target substances of PFASs. .

[0020] Table 2 lists the internal standard compounds corresponding to the target substances of OPEs. .

[0021] Preparation of placental freeze-dried powder: Placentas collected from the hospital were cut into 5 cm pieces. 2 Small fragments were placed in a petri dish and frozen in a refrigerator at -20 to -45°C for more than 24 hours. After freezing, they were placed in a vacuum dryer to dry until they were free of moisture. After drying, they were placed in a mortar and ground into powder for later use.

[0022] Sample blank: Weigh 0.5 g of lyophilized placenta powder, add 2 ng (20 μL of 100 ppb) of PFASs and OPEs internal standards, add 6 ml of acetonitrile (protein remover), vortex for 2 min, centrifuge at 6000 rpm for 30 min, extract the supernatant, add another 6 ml of acetonitrile, and repeat the above steps. Combine the two supernatants and perform solid-phase extraction on the combined supernatant using a phospholipid column (CNWBOND phospholipid-removing PPR Pro, 300 mg 3 mL). Collect the extracted solution, concentrate to near dryness under nitrogen at 30℃, redissolve in 200 μL of methanol, and analyze by LC-MS. Sample and standard addition: Weigh 0.5 g of placental lyophilized powder, add 2 ng (20 μL of 100 ppb) of PFASs and OPEs standards (Wellington Canada, Alta China), then add 2 ng (20 μL of 100 ppb) of PFASs and OPEs internal standards, add 6 ml of acetonitrile (protein removal agent), vortex for 2 min, centrifuge at 6000 rpm for 30 min, extract the supernatant, add 6 ml of acetonitrile, repeat the above steps, combine the two supernatants, and perform solid-phase extraction on the combined supernatant using a phospholipid column (CNWBOND phospholipid-free PPR Pro, 300 mg 3 mL). Collect the extracted solution, concentrate to near dryness under nitrogen at 30℃, redissolve in 200 μL of methanol, and analyze by LC-MS. The LC-MS analysis conditions are as follows: PFASs sample analysis was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) with Hypersil... Sample separation was performed using a GOLD liquid chromatography column (Thermo Fisher Scientific, 1.5 μm, 2.1 x 100 mm). Mobile phase A (aqueous phase) and mobile phase B (methanol phase) both contained 2 mmol / L ammonium acetate and 2 mmol / L N-methylpiperidine. The specific elution process is shown in Tables 3 and 4. The column temperature was set to 40℃, and the injection plate temperature was set to 8℃. Mass spectrometry analysis was performed using electrospray ionization (ESI-) for PFASs in negative ion mode and positive ion mode (ESI+) for OPEs. Full scan (Full MS) was coupled with data-dependent mass spectrometry (dd-MS2), with a scan range of m / z 100–1000. The test results are as follows: Figure 1 , 2 As shown, Figure 1 The recovery test results for PFASs Figure 2 The recovery rate test results for OPEs show that all 17 added PFASs were recovered, with 14 of them having a recovery rate of 100%–130%, indicating excellent recovery performance. However, for carboxylic acid compounds, the recovery performance of ultra-long-chain PFASs was poor due to their longer chains and instability. All 22 added OPEs were recovered, with 10 of them having a recovery rate of 100%–130%, indicating excellent recovery performance. Except for BEHP and TEHP, the recovery rates of the remaining substances were also above 50%, meeting the detection requirements.

[0023] Table 3 PFASs elution procedure .

[0024] Table 4 OPEs Elution Procedure .

[0025] Comparative Example 1 The difference from Example 1 is that methanol is used as the protein removal agent, that is, acetonitrile is replaced with methanol. The rest of the steps are the same as in Example 1. However, it cannot completely remove the protein. White particles can be clearly observed in the supernatant, which will cause column blockage and damage to the instrument in subsequent instrument detection.

[0026] Comparative Example 2 Replacing the dephospholipid column (CNWBOND Dephospholipid PPR Pro, 300 mg 3 mL) with one that does not use a dephospholipid column, while keeping the rest of the steps the same as in Example 1, failed to remove the fat. Yellow oily substances were observed in the solution, indicating that not using a dephospholipid column would result in fat residue in the sample, which would subsequently interfere with and damage the instrument.

[0027] The technical solution disclosed in this invention utilizes acetonitrile to remove proteins from biological samples, followed by further extraction of lipids from the sample using a phospholipid column to ensure that the target analyte is not interfered with. Nitrogen concentration and methanol reconstitution improve sample purity and detection sensitivity. Recovery test results show that the recoveries of PFASs and OPEs both reach the ideal range, verifying the reliability and effectiveness of the method. Therefore, this method simultaneously extracts PFASs and OPEs, offering advantages such as relatively low cost, short processing time, simple operation, and good experimental results. Furthermore, because samples are difficult to obtain, a single sample can be used to detect both substances simultaneously, resulting in more efficient results. This invention provides a powerful means for the simultaneous extraction and detection of different compounds in biological samples.

[0028] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the implementation. The scope of protection of the present invention should be determined by the scope defined in the claims. Other variations or modifications can be made based on the above description. Obvious variations or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A pretreatment method for perfluorinated and polyfluoroalkyl compounds and organophosphates in biological samples, characterized in that, The pretreatment method includes the following steps: adding acetonitrile and internal standard to the biological sample, vortexing and centrifuging, extracting the supernatant, adding acetonitrile again, vortexing and centrifuging, extracting the supernatant again, combining the two supernatants, performing solid-phase extraction on the combined supernatant using a phospholipid column, concentrating with nitrogen, redissolving with methanol, and analyzing the redissolved sample.

2. The pretreatment method according to claim 1, characterized in that, The mass ratio of the biological sample to acetonitrile is 0.5~1:4.5~6.5, preferably 1:4.

7.

3. The pretreatment method according to claim 1, characterized in that, The biological sample was a cold-dried placental sample.

4. The pretreatment method according to claim 1, characterized in that, The vortex lasts for 1 to 2 minutes.

5. The pretreatment method according to claim 1, characterized in that, The centrifugation conditions are as follows: The centrifuge speed is 5000~6000 rpm; The centrifugation time is 10-20 minutes.

6. The pretreatment method according to claim 1, characterized in that, The phospholipid columns were CNWBOND dephospholipid PPR Pro, 300 mg 3 mL.

7. The pretreatment method according to claim 1, characterized in that, The nitrogen concentration temperature is 30~40℃; The nitrogen concentration time is 3-4 hours. Mobile phase A (aqueous phase) and mobile phase B (methanol phase) both contain 2 mmol / L ammonium acetate and 2 mmol / L N-methylpiperidine. The PFASs elution process is as follows: the flow rate is 0.3 mL / min for both phases. 30%B, 70%A, 0~1.5 min; 50%B, 50%A, 1.5 min; 60%B, 40%A, 6.5 min; 75%B, 25%A, 20.5min; 85%B, 15%A, 23.5 min; 100%B, 0%A, 24 min; 0%B, 100%A, 29 min; 30%B, 70%A, 29.1~35.6min; The OPEs elution process is as follows: the flow rate is 0.4 mL / min; 30%B, 70%A, 0~1 min; 50%B, 50%A, 1 min; 100%B, 0%A, 9 min; 0%B, 100%A, 16.8 min; 30%B, 70%A, 17~20 min; The column temperature was set to 40℃, and the injection plate temperature was set to 8℃. Electrospray ionization was used for PFASs in negative ion mode (ESI-) and OPEs in positive ion mode (ESI+). Full scan and data-dependent mass spectrometry were used together, with the full scan range being 100~1000 m / z.

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