Method for extracting organic nitrate ester from pine needles under biological passive sampling technology

Abstract

The invention relates to the technical field of analytical chemistry sample pretreatment, and discloses a method for extracting organic nitrate in pine needles under a biological passive sampling technology, which comprises the following steps: after methanol ultrasonic extraction and primary centrifugation, setting a pre-purification step: placing supernate in a low-temperature environment of-25 DEG C to-35 DEG C for standing treatment, by utilizing the characteristic that the solubility of matrix interferents such as vegetable wax and lipid is reduced at the low temperature, the matrix interferents are separated out in a solid form; and separating and removing the solidified interferent through secondary centrifugation to obtain a clear supernatant, and then carrying out solid-phase extraction and purification. Through the pre-purification step, the blockage of the solid-phase extraction column is effectively avoided, the recovery rate of the target object is remarkably improved, and the matrix effect in the analysis test is weakened, so that the accuracy and precision of the method are improved.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry sample pretreatment technology, specifically to a method for extracting organic nitrates from pine needles using biological passive sampling technology. Background Technology

[0002] Pine needles, due to their surface properties, are widely used as biological passive samplers to enrich semi-volatile organic compounds in the atmosphere, among which organic nitrates are an important class of substances that require attention in environmental monitoring. Accurate quantitative analysis of organic nitrates in pine needle samples is a crucial step in assessing the regional atmospheric environmental conditions.

[0003] Existing analytical procedures typically include solvent extraction, purification and enrichment, and instrumental analysis. In the extraction stage, polar organic solvents such as methanol are commonly used to dissolve the target compound from the pine needle matrix via methods such as ultrasound. In the purification stage, solid-phase extraction (SPE) technology is widely used due to its ease of operation and high efficiency, for separating and enriching target organic nitrates from complex extracts.

[0004] However, in practical applications, the above-mentioned technical solutions have inherent drawbacks. Pine needles, as a complex biological matrix, contain not only the target analyte but also a large number of endogenous compounds such as plant waxes, lipids, and pigments. During solvent extraction, solvents such as methanol, while dissolving the target organic nitrates, inevitably dissolve a large amount of these highly interfering matrix components.

[0005] When crude extracts containing high concentrations of interfering matrix components are directly used for solid-phase extraction (SPE) purification, a series of problems arise. Plant waxes and lipid compounds readily precipitate and aggregate on the surface or within the pores of the SPE column packing material, leading to physical blockage. This results in extremely slow or even complete cessation of sample flow through the column, prolonging pretreatment time and hindering automation and high-throughput processing. Furthermore, even without severe blockage, these highly interfering matrix components compete with the target analyte for active sites on the SPE packing material, affecting effective adsorption and elution, thus reducing the recovery rate and stability of the target organic nitrate.

[0006] More importantly, when incompletely purified sample solutions enter sophisticated analytical instruments such as high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), residual matrix interferences compete with the target analyte for ionization efficiency in the ion source, producing a significant matrix effect, typically manifested as signal suppression. This signal suppression severely impacts the sensitivity and accuracy of the analysis, significantly reducing the reliability of quantitative results. Therefore, current techniques lack an effective pre-purification step for the extract before solid-phase extraction, failing to reliably and efficiently remove specific strong matrix interferences, thus limiting the accuracy and reliability of the entire analytical method. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for extracting organic nitrates from pine needles using biological passive sampling technology. This method solves the problem that existing technologies lack an effective pre-purification step for the extract before solid-phase extraction, which makes it impossible to stably and efficiently remove specific strong matrix interferences.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting organic nitrates from pine needles using a biological passive sampling technique, comprising the following steps: S1. The freeze-dried pine needle sample was extracted with methanol by ultrasonic extraction to obtain the extract; S2. Perform primary centrifugation on the extract and collect the supernatant; S3. Place the supernatant in a low-temperature environment for static treatment to allow matrix interferences to precipitate out. S4. Centrifuge the supernatant after it has been allowed to stand for a second time to separate and remove the precipitated matrix interferences and obtain a clear supernatant. S5. The clarified supernatant is purified by solid-phase extraction, and the purified sample is concentrated and reconstituted.

[0009] Preferably, in step S3, the temperature of the low-temperature environment is between -25°C and -35°C, and the settling time is 2.5 to 3.5 hours. Through a specific combination of temperature and time, specific matrix interferences dissolved in the methanol extract can reach a supersaturated state and be fully extracted.

[0010] Preferably, in step S1, the ultrasonic extraction temperature is maintained at 14-16°C. This temperature range helps to control the thermal stability of the target material while ensuring extraction efficiency.

[0011] Preferably, the temperature of the primary centrifugation in step S2 and the secondary centrifugation in step S4 is 3-5°C. Low-temperature centrifugation helps maintain the solid form of the precipitated matrix interferences, which is beneficial for their sedimentation and separation.

[0012] Preferably, the matrix interference substances precipitated in step S3 are plant waxes and lipid compounds.

[0013] Preferably, the solid-phase extraction purification in step S5 specifically involves: using a solid-phase extraction column to adsorb the clarified supernatant and eluting it with methanol.

[0014] Preferably, the resolution in step S5 specifically involves resolution using a methanol solution containing tropidine and pinocembryonic acid as internal standards.

[0015] Preferably, in the methanol solution containing the internal standard, the concentration of tropinone is 1.8–2.2 mg / L and the concentration of pinocembranoic acid is 3.8–4.2 mg / L.

[0016] Preferably, before step S1, the step further includes a freeze-drying step of the pine needle sample, wherein the freeze-drying pressure is 10-15 mbar and the temperature is -55°C to -65°C.

[0017] Preferably, in step S1, the amount of pine needle sample used is 9.5 to 10.5 g, and the sample is pre-treated into small segments with a length of 0.8 to 1.2 cm.

[0018] This invention provides a method for extracting organic nitrates from pine needles using a passive biological sampling technique. It offers the following advantages: 1. By performing low-temperature settling and secondary centrifugation before the solid-phase extraction step, this invention effectively separates and removes a large number of high molecular weight or highly hydrophobic matrix interferences. This directly reduces the risk of physical blockage of the subsequent solid-phase extraction column or irreversible adsorption and contamination of the active sites on the packing surface, thereby ensuring the smoothness and stability of the purification process when multiple samples are processed continuously.

[0019] 2. This invention utilizes the physical property that the solubility of matrix interfering substances such as plant waxes and lipids in methanol solution is significantly reduced in this temperature range by setting a low-temperature settling step of -25°C to -35°C, causing them to precipitate out of the extract in solid form. The subsequent secondary centrifugation step effectively separates these solidified interfering substances, thereby achieving targeted removal of these specific strong matrix interfering substances and improving the efficiency of subsequent purification steps.

[0020] 3. The present invention improves the matrix purity of the final sample solution obtained. During chromatography-mass spectrometry analysis, it reduces the signal suppression effect during ionization caused by residual matrix interferences, i.e., the matrix effect. This makes the signal response of the target analyte more stable, thereby improving the accuracy of qualitative identification and quantitative calculation of organic nitrates. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Example: This embodiment provides a method for extracting organic nitrates from pine needles according to the present invention, the specific steps of which are as follows: Sample pretreatment: The collected pine needle samples were freeze-dried. The freeze dryer pressure was set to 12 mbar, the cold trap temperature was set to -60℃, and the drying time was 24 hours.

[0023] Sample crushing and weighing: The freeze-dried pine needle sample was cut into small segments of 1.0 cm in length using stainless steel scissors, and 10.0 g was accurately weighed using an analytical balance.

[0024] Ultrasonic extraction: Place the weighed sample in an extraction container and add 30 ml of methanol. Place the container in a constant temperature ultrasonic instrument, set the water bath temperature to 15°C, and perform ultrasonic extraction for 15 minutes. Repeat this process three times, and combine all three extracts.

[0025] Primary centrifugation: Centrifuge the combined extracts at 8000 rpm for 10 minutes at 4°C and collect the supernatant.

[0026] Low-temperature settling treatment: Transfer the supernatant obtained in step 4 to a clean container, seal it, and place it in a low-temperature environment of -30°C for 3 hours.

[0027] Secondary centrifugation: The liquid that has been subjected to low-temperature settling is immediately centrifuged at 8000 rpm for 10 minutes at 4°C to separate and remove the precipitated solid precipitate, and obtain a clear supernatant.

[0028] Solid-phase extraction purification: Pass the clear supernatant obtained in step 6 through a solid-phase extraction column pre-activated with 10 ml of methanol. After all the sample has passed through, elute with 10 ml of methanol and collect the eluent.

[0029] Concentration and redissolution: The collected eluent was concentrated to near dryness using a nitrogen blower, and then 300 µL of a methanol solution containing 2.0 mg / L tropidine and 4.0 mg / L pinocemetic acid was precisely added for redissolution to obtain the sample solution to be tested.

[0030] Example 2 This embodiment provides a method for extracting organic nitrates from pine needles according to the present invention, the specific steps of which are as follows: Sample pretreatment: The collected pine needle samples were freeze-dried. The freeze dryer pressure was set to 10 mbar, the cold trap temperature was set to -55℃, and the drying time was 22 hours.

[0031] Sample crushing and weighing: The freeze-dried pine needle sample was cut into small segments of 0.8 cm in length using stainless steel scissors, and 9.5 g was accurately weighed using an analytical balance.

[0032] Ultrasonic extraction: Place the weighed sample in an extraction container and add 30 ml of methanol. Place the container in a constant temperature ultrasonic instrument, set the water bath temperature to 14℃, and perform ultrasonic extraction for 14 minutes. Repeat this process 3 times, and combine all 3 extracts.

[0033] Primary centrifugation: Centrifuge the combined extracts at 8000 rpm for 8 minutes at 3°C ​​and collect the supernatant.

[0034] Low-temperature settling treatment: Transfer the supernatant obtained in step 4 to a clean container, seal it, and place it in a low-temperature environment of -25°C for 2.5 hours.

[0035] Secondary centrifugation: The liquid that has been subjected to low-temperature settling is immediately centrifuged at 8000 rpm for 8 minutes at 3°C ​​to separate and remove the precipitated solid precipitate, and obtain a clear supernatant.

[0036] Solid-phase extraction purification: Pass the clear supernatant obtained in step 6 through a solid-phase extraction column pre-activated with 9 ml of methanol. After all the sample has passed through, elute with 9 ml of methanol and collect the eluent.

[0037] Concentration and redissolution: The collected eluent was concentrated to near dryness using a nitrogen blower, and then 280 µL of a methanol solution containing 1.8 mg / L tropidine and 3.8 mg / L pinocemetic acid was precisely added for redissolution to obtain the sample solution to be tested.

[0038] Example 3 This embodiment provides a method for extracting organic nitrates from pine needles according to the present invention, the specific steps of which are as follows: Sample pretreatment: The collected pine needle samples were freeze-dried. The freeze dryer pressure was set to 15 mbar, the cold trap temperature was set to -65℃, and the drying time was 26 hours.

[0039] Sample crushing and weighing: The freeze-dried pine needle sample was cut into small segments of 1.2 cm in length using stainless steel scissors, and 10.5 g was accurately weighed using an analytical balance.

[0040] Ultrasonic extraction: Place the weighed sample in an extraction container and add 30 ml of methanol. Place the container in a constant temperature ultrasonic instrument, set the water bath temperature to 16℃, and perform ultrasonic extraction for 16 minutes. Repeat this process 3 times, and combine all 3 extracts.

[0041] Primary centrifugation: Centrifuge the combined extracts at 8000 rpm for 12 minutes at 5°C and collect the supernatant.

[0042] Low-temperature settling treatment: Transfer the supernatant obtained in step 4 to a clean container, seal it, and place it in a low-temperature environment of -35°C for 3.5 hours.

[0043] Secondary centrifugation: The liquid that has been subjected to low-temperature settling is immediately centrifuged at 8000 rpm for 12 minutes at 5°C to separate and remove the precipitated solid precipitate, and obtain a clear supernatant.

[0044] Solid-phase extraction purification: Pass the clear supernatant obtained in step 6 through a solid-phase extraction column pre-activated with 11 ml of methanol. After all the sample has passed through, elute with 11 ml of methanol and collect the eluent.

[0045] Concentration and redissolution: The collected eluent was concentrated to near dryness using a nitrogen blower, and then 320 µL of a methanol solution containing 2.2 mg / L tropidine and 4.2 mg / L pinocemetic acid was precisely added for redissolution to obtain the sample solution to be tested.

[0046] Comparative Example Comparative Example 1: Compared with Example 1, the difference is that after the primary centrifugation in step 4, the obtained supernatant was directly used for solid-phase extraction purification in step 7, omitting the low-temperature settling treatment in step 5 and the secondary centrifugation in step 6. All other steps are the same.

[0047] Comparative Example 2: Compared with Example 1, the difference is that the temperature of the low-temperature standing treatment in step 5 is not -30°C, but is carried out in a low-temperature environment of 4°C, while the rest are the same.

[0048] Comparative Example 3: Compared with Example 1, the difference is that the low-temperature standing treatment time in step 5 is shortened to 30 minutes instead of 3 hours, while the rest are the same.

[0049] Comparative Example 4: Compared with Example 1, the difference is that after the low-temperature standing treatment in step 5, the supernatant was carefully transferred by pouring for subsequent solid-phase extraction purification, and the second centrifugation step in step 6 was omitted, while the rest were the same.

[0050] Test Example 1: Solid Phase Extraction Column Flux Stability Test Experimental steps S1. Take 30 ml of the supernatant from each of Examples 1-3 and Comparative Examples 1-4 before entering the solid-phase extraction step. For Examples 1-3 and Comparative Examples 2-3, this is the supernatant after the second centrifugation; for Comparative Example 1, this is the supernatant after the first centrifugation; for Comparative Example 4, this is the supernatant poured out after standing at low temperature.

[0051] S2. Prepare 8 Waters solid-phase extraction columns of the same model and batch. Activate each column with the corresponding volume of methanol, as described in the examples and comparative examples.

[0052] S3. Add the 8 different supernatants prepared in step S1 to the corresponding activated solid phase extraction columns and start timing immediately.

[0053] S4. Record the time taken for each supernatant to completely descend to the surface of the packing material from the initial addition of the supernatant. Set an upper limit of 20 minutes; if the liquid has not completely passed through within this time, record it as slow flow or blockage.

[0054] S5. Observe and record the final state of each column, including "complete", "slow flow" or "blocked".

[0055] Experimental data Solid phase extraction column flux stability test results Results and Analysis According to the test data in Table 1, the clear supernatants obtained using the methods of Examples 1, 2, and 3 all passed smoothly through the solid-phase extraction column within 14 minutes. In contrast, the liquids obtained using the methods of Comparative Examples 1, 2, 3, and 4 all exhibited significantly reduced flow rates or complete blockage when passing through the solid-phase extraction column, failing to complete the loading process within the set 20 minutes.

[0056] The mechanism behind the above phenomena lies in the fact that the method in Comparative Example 1 lacked the low-temperature settling and secondary centrifugation steps. This resulted in a large amount of matrix interference substances, such as plant waxes and lipids dissolved in the extract, directly contacting the packing material when entering the solid-phase extraction column, causing physical blockage of the column's internal channels. The low-temperature conditions in Comparative Examples 2 and 3 were insufficient to fully precipitate the matrix interference substances in the methanol solution, leaving a large amount of interference substances remaining in the supernatant, thus causing similar blockage problems. Although Comparative Example 4 involved low-temperature settling, the secondary centrifugation step was omitted, causing the precipitated solid interference substances to be reintroduced into the liquid during subsequent pouring and transfer, similarly leading to blockage of the solid-phase extraction column.

[0057] The method disclosed in this invention utilizes the physical property that the solubility of matrix interfering substances in methanol solution decreases sharply in this specific temperature range (-25°C to -35°C), causing them to precipitate from the liquid phase in solid form. The subsequent secondary centrifugation step separates and removes these solidified matrix interfering substances from the supernatant based on density differences. This specific combination of steps significantly reduces the concentration of compounds that could cause blockage in the final clarified supernatant entering the solid-phase extraction column, thereby maintaining the smoothness of the purification process.

[0058] Test Example 2: Recovery Test of Target Organic Nitrate Esters Experimental steps S1. Take a blank pine needle sample that has been freeze-dried and homogenized. This sample has been pre-determined to be free of the target organic nitrate ester. Divide the blank sample into 8 equal portions, each weighing 10.0g.

[0059] S2. Add the same volume and concentration of mixed organic nitrate standard solution to each blank sample, so that the theoretical spiking concentration of each target analyte in each sample is 50.0 µg / L.

[0060] S3. These 8 spiked samples were extracted, purified and reconstituted according to the complete methods described in Examples 1-3 and Comparative Examples 1-4 to obtain the final test sample solution.

[0061] S4. Using a high-performance liquid chromatography-tandem mass spectrometry system, analyze each final sample solution to determine the actual concentration of a representative organic nitrate.

[0062] S5. Calculate the spiked recovery rate according to the formula: Spike recovery rate (%) = (Measured concentration / Theoretical spiked concentration) × 100%.

[0063] Experimental data Recovery test results of target organic nitrate ester spiked Results and Analysis Based on the test data, the recoveries of the target analytes obtained by processing the spiked samples using the methods of Examples 1, 2, and 3 ranged from 89.6% to 94.2%. In contrast, the recoveries of the target analytes obtained using the methods of Comparative Examples 1, 2, 3, and 4 ranged from 67.4% to 80.2%, all of which were lower than those in the Example groups.

[0064] In the treatments of Comparative Examples 1, 2, 3, and 4, the low recovery rates were directly related to the incomplete removal of matrix interferences. Because these methods failed to effectively remove compounds such as plant waxes and lipids from the solution, these interferences compete with the target analytes for active sites on the packing material during the solid-phase extraction step, or directly cause physical blockage. This results in some target analytes not being effectively adsorbed or being lost along with the liquid during the blockage process, thus reducing the final recovery rate.

[0065] The method disclosed in this invention introduces a specific low-temperature settling step, which causes specific matrix interferences dissolved in the methanol extract to precipitate due to reduced solubility. These solid-phase interferences are then removed from the system through a subsequent secondary centrifugation step. This pre-purification treatment significantly reduces the concentration of interferences in the liquid entering the solid-phase extraction step, ensuring that the target organic nitrate can efficiently interact with and be enriched by the solid-phase extraction packing material, ultimately resulting in a higher spike recovery rate.

[0066] Test Example 3: Evaluation of Sample Matrix Effect Experimental steps S1. Take a homogeneous blank pine needle sample that has been pre-determined to be free of organic nitrate esters, and divide it into 8 equal portions, each weighing 10.0g.

[0067] S2. These 8 blank samples were extracted, purified and reconstituted according to the complete methods described in Examples 1-3 and Comparative Examples 1-4 to obtain 8 different final matrix solutions.

[0068] S3. Prepare an organic nitrate standard solution using pure methanol as the solvent, with a concentration of 50.0 µg / L. This is a pure solvent standard solution.

[0069] S4. Take the 8 final matrix solutions prepared in step S2 and add organic nitrate ester mixed standards to them so that the final concentration of the target substance in the matrix solution is also 50.0 µg / L.

[0070] S5. Using a high-performance liquid chromatography-tandem mass spectrometry system, the signal response intensity of the target analytes in the pure solvent standard solution and the eight matrix solutions was determined.

[0071] S6. Calculate the matrix effect according to the formula: Matrix effect (%) = (Signal response intensity of the target analyte in the matrix solution / Signal response intensity of the target analyte in the pure solvent standard solution) × 100%.

[0072] Experimental data Sample matrix effect evaluation results Results and Analysis Test data show that the matrix effect of the sample solutions prepared using the methods of Examples 1, 2, and 3 is in the range of 90.8% to 95.2%. In contrast, the matrix effect of the sample solutions prepared using the methods of Comparative Examples 1, 2, 3, and 4 is in the range of 58.6% to 72.4%, indicating significant signal suppression.

[0073] The signal suppression observed in the comparative examples is due to the high concentration of matrix interferences remaining in the final sample solution. These interferences compete with the target analyte for the energy or space required for ionization when entering the ion source of the mass spectrometer, thus reducing the ionization efficiency of the target analyte and weakening its signal response. The methods in Comparative Examples 1-4, lacking crucial low-temperature processing or separation steps, or using conditions insufficient to effectively remove interferences, retained a large amount of matrix components, resulting in a significant matrix effect.

[0074] The method disclosed in this invention, by introducing a low-temperature settling step at -25°C to -35°C, combined with a subsequent secondary centrifugation, can effectively remove strong matrix interfering substances such as plant waxes and lipids from the methanol extract based on differences in physical solubility. This specific combination of steps significantly improves the purity of the final sample solution, resulting in a substantial reduction in the concentration of interfering substances entering the analytical instrument. Therefore, the ionization process of the target organic nitrate ester is less affected, and its signal response intensity approaches the level in the pure solvent, thus providing a basis for obtaining accurate quantitative analysis results.

[0075] Test Example 4: Method Precision Test Experimental steps S1. Take pine needle samples from the same batch containing endogenous organic nitrates, freeze-dry and homogenize them.

[0076] S2. Accurately weigh multiple portions of the homogenized sample, each 10.0 g. Prepare 6 parallel samples for each method in Examples 1-3 and Comparative Examples 1-4.

[0077] S3. Extract, purify and reconstitute the 6 parallel samples from each group according to the complete methods described in Examples 1-3 and Comparative Examples 1-4 to obtain the final sample solution to be tested.

[0078] S4. Using a high-performance liquid chromatography-tandem mass spectrometry system, analyze all the final test sample solutions to determine the concentration of the same endogenous organic nitrate.

[0079] S5. For the measurement results of 6 parallel samples in each group, calculate the mean concentration and relative standard deviation.

[0080] Experimental data Method precision test results Results and Analysis Test data show that the relative standard deviations (RSDs) of the measurement results obtained by processing parallel samples using the methods of Examples 1, 2, and 3 ranged from 3.9% to 5.1%. In contrast, the RSDs of the results obtained using the methods of Comparative Examples 1, 2, 3, and 4 ranged from 13.8% to 18.5%. The RSD values ​​of the Example groups were significantly lower than those of the Comparative Examples.

[0081] The high relative standard deviations observed in the comparative examples reflect insufficient method reproducibility, fundamentally due to the instability of the matrix interference removal process. In Comparative Example 1, the lack of cryogenic treatment meant that matrix interferences in different parallel samples might interact with the solid-phase extraction packing material in a non-uniform manner, leading to random analyte loss. In Comparative Examples 2 and 3, insufficient cryogenic conditions resulted in incomplete and unstable precipitation of matrix interferences, causing differences in the final matrix levels entering the analyzer for each parallel sample. In Comparative Example 4, omitting the second centrifugation caused the precipitated solid interferences to be carried into subsequent steps in random quantities, directly introducing operational errors.

[0082] The method disclosed in this invention ensures that target matrix interferences are stably and consistently converted from methanol solution to solid precipitates in different parallel samples by performing a low-temperature settling step at -25°C to -35°C. The subsequent secondary centrifugation step provides a standardized mechanical separation process, enabling reproducible removal of these solid-phase interferences from the liquid phase. This specific combination of steps guarantees a high degree of pre-purification for each parallel sample, allowing the entire analytical process, including solid-phase extraction and instrumental analysis, to be performed against a stable and predictable matrix background, ultimately resulting in low relative standard deviation and high method precision.

[0083] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting organic nitrates from pine needles using a biological passive sampling technique, characterized in that, Includes the following steps: S1. The freeze-dried pine needle sample was extracted with methanol by ultrasonic extraction to obtain the extract; S2. Perform primary centrifugation on the extract and collect the supernatant; S3. Place the supernatant in a low-temperature environment for static treatment to allow matrix interferences to precipitate out. S4. Centrifuge the supernatant after it has been allowed to stand for a second time to separate and remove the precipitated matrix interferences and obtain a clear supernatant. S5. The clarified supernatant is purified by solid-phase extraction, and the purified sample is concentrated and reconstituted.

2. The method for extracting organic nitrates from pine needles using a biological passive sampling technique according to claim 1, characterized in that, In step S3, the temperature of the low-temperature environment is between -25°C and -35°C, and the settling time is 2.5 to 3.5 hours.

3. The method for extracting organic nitrates from pine needles using a biological passive sampling technique according to claim 1, characterized in that, In step S1, the temperature of ultrasonic extraction is maintained at 14-16℃.

4. The method for extracting organic nitrates from pine needles using a biological passive sampling technique according to claim 1, characterized in that, The temperature of the primary centrifugation in step S2 and the secondary centrifugation in step S4 is 3-5℃.

5. The method for extracting organic nitrates from pine needles using a biological passive sampling technique according to claim 1, characterized in that, The matrix interference substances precipitated in step S3 are plant waxes and lipid compounds.

6. The method for extracting organic nitrates from pine needles using a biological passive sampling technique according to claim 1, characterized in that, The solid-phase extraction purification in step S5 specifically involves: using a solid-phase extraction column to adsorb the clarified supernatant and eluting it with methanol.

7. The method for extracting organic nitrates from pine needles using a biological passive sampling technique according to claim 1, characterized in that, The reconstitution in step S5 specifically involves reconstitution using a methanol solution containing tropidine and pinocembryonic acid as internal standards.

8. The method for extracting organic nitrates from pine needles using a biological passive sampling technique according to claim 7, characterized in that, In the methanol solution containing the internal standard, the concentration of tropinone is 1.8–2.2 mg / L and the concentration of pinocembrin is 3.8–4.2 mg / L.

9. The method for extracting organic nitrates from pine needles using a biological passive sampling technique according to claim 1, characterized in that, Before step S1, the method further includes a freeze-drying step of the pine needle sample, wherein the freeze-drying pressure is 10-15 mbar and the temperature is -55℃ to -65℃.

10. The method for extracting organic nitrates from pine needles using a biological passive sampling technique according to claim 1, characterized in that, In step S1, the amount of pine needle sample used is 9.5 to 10.5 g, and the sample is pre-treated into small segments with a length of 0.8 to 1.2 cm.

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