A method for detecting phthalate monoester compound residues in crop planting environment samples

Abstract

The present invention provides a method for detecting residual phthalate compounds in samples from crop planting environments, comprising: (1) extraction: pre-treating samples from the crop planting environment, adding an isotope internal standard, and extracting with an extracting solution; (2) concentration and enrichment: concentrating the extracting solution and enriching the target compound through an SPE column; and (3) detection: detecting with HPLC-MS / MS. The present invention finds that phthalate esters are easily converted into phthalate monoesters in an agricultural environment, provides an HPLC-MS / MS analysis method, and gives HPLC-MS / MS spectra of 19 typical phthalate monoester compounds, confirming the spectrum of phthalate monoester compounds remaining in soil and five kinds of vegetables. Compared with direct detection of phthalates, detection of phthalate monoester compounds can more accurately assess the ecological and health risks posed by plasticizers to agricultural planting systems.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis and detection of environmental samples, and in particular to a method for detecting residual plasticizer degradation products in crop planting environment samples, and in particular to a method for detecting residual phthalate monoester compounds in crop planting environment samples. Background Art

[0002] Phthalate esters (PAEs), also known as phthalic acid esters, are a class of important chemical raw materials and a major industrial commodity. According to relevant statistics, global production of phthalates reaches 6 million tons annually. They have a wide range of uses, found in nearly all industrial products, including pesticides, fertilizers, cosmetics, pharmaceuticals, lubricants, and coatings. However, they are primarily used as additives in plastics to increase their plasticity, flexibility, and strength, hence their nickname of plasticizer. They are added in amounts as high as 20%-60% in some products. Because PAEs are non-chemically bonded to the polyolefin plastic matrix, they are easily released and can migrate and diffuse from the plastic into environmental media such as soil, water, and air. Plastic greenhouses are a key form of protected agriculture. Using them for vegetable cultivation offers advantages such as high yields and minimal seasonal impact, playing a significant role in alleviating the supply and demand imbalance during the vegetable off-season. The extensive use of plastic greenhouses and plastic films has weakened the hydrogen bonds or van der Waals forces between PAEs and plastic molecules over time, making them easily released and migrated into the greenhouse air, irrigation water, or settled and absorbed by the soil, increasing the content of PAEs in the soil. Since PAEs are semi-volatile organic compounds, there are two main views on the ways in which vegetables in plastic greenhouses absorb and accumulate PAEs: (1) PAEs in the soil are absorbed by plant roots and transported to stems and leaves. PAEs plasticizers can pass through the root cortex and enter the xylem, accumulate in plant stems through the action of root hair cells, or reach leaves and accumulate through transport; (2) Due to the low Henry coefficient, high boiling point and low vapor pressure of PAEs, they can only evaporate slowly into the atmosphere. PAEs existing in the atmosphere will be strongly adsorbed on particulate matter and then absorbed and accumulated by the stems and leaves of greenhouse plants.

[0003] Currently, PAEs have been detected in soil, air, water, and agricultural products. Due to their stability, long-lasting persistence, and strong bioaccumulative toxicity, they pose significant risks to humans and the environment, and are therefore listed as priority pollutants. Studies have shown that PAEs can have endocrine disrupting effects on experimental animals, produce reproductive and developmental toxicity, immunotoxicity, and neurotoxicity, and have teratogenic, carcinogenic, and mutagenic effects. The most concerning and serious hazards are their endocrine disrupting effects and their impact on the reproductive system development of male animals. The U.S. Environmental Protection Agency (EPA) has designated six phthalates, including dimethyl phthalate (DMP), diethyl phthalate (DEP), and dibutyl phthalate (DBP), as "priority pollutants." my country has also designated DMP, DBP, and di(2-ethylhexyl) phthalate (DEHP) as priority pollutants for monitoring. Numerous studies have been conducted on phthalate detection methods both domestically and internationally. CN20151059787.9 uses a gas chromatography-tandem mass spectrometry (GC-MS / MS) analysis method to simultaneously detect multiple plasticizers. CN20191085764.2 provides a method for detecting phthalates in vegetables grown in contaminated soil, comprising (1) crude sample extraction; (2) sample extraction; (3) sample purification; (4) sample elution; and (5) detection. After extraction and adsorption treatment using a specific photosensitive adsorption filler, phthalate detection is achieved by chromatography-mass spectrometry.

[0004] Due to the readily hydrolyzed nature of diester compounds, phthalates in agricultural environments are readily converted to phthalate monoesters through hydrolysis by soil microorganisms and plants. Phthalate monoesters exhibit a variety of toxic effects, including endocrine disruption, reproductive and developmental toxicity, and neurotoxicity, and can even cause teratogenicity and carcinogenesis. Compared to the parent phthalate compounds, metabolites such as phthalate monoesters more readily penetrate the phospholipid bilayer barrier of cell membranes, resulting in more potent biological toxicity. Although the health toxicity and ecotoxicology of phthalate monoesters are still underdeveloped, some of these compounds, such as mono(2-ethylhexyl)phthalate, monobutyl phthalate, monomethyl phthalate, and monobenzyl phthalate, have been included in the EDSP (Endocrine Disruptor Screening Program) Universe of Chemicals priority screening list of pollutants due to their potent endocrine disrupting effects. Studies have estimated the contribution of different exposure pathways to the total human exposure to phthalate monoesters and found that dietary exposure in crops accounts for approximately 70%. Therefore, there is an urgent need to develop detection methods for phthalate monoesters in crop cultivation systems to clarify the accumulation characteristics and doses of these compounds in different crops.

[0005] For the detection of phthalate monoester chemicals, current research mainly focuses on the detection methods of phthalate metabolites in human urine, human hair or nail samples (for example, CN20221052117.0). So far, no analytical method for phthalate monoester compounds in crop planting systems has been established. Compared with human samples, soil and crop samples usually contain complex components such as soil organic matter and chlorophyll that interfere with instrument response. The above-mentioned wide variety of interfering components increases the difficulty of extracting and purifying the target compounds in the sample, thereby interfering with the detection and analysis of the instrument. In addition, due to factors such as the large amount of use and the multiple potential sources of this type of chemical, it is widely distributed in a variety of environmental media, and the quality control requirements for the entire analysis process of soil and crop sample collection-pretreatment process-instrument analysis are high. Some commonly used phthalate monoesters are isomers with the same parent ion and daughter ion, which places high requirements on instrument chromatographic separation. In order to effectively assess the potential ecological risks of phthalate monoester compounds in soil and the potential health risks of such compounds through crop dietary exposure, the invention of an analytical method that can simultaneously accurately quantify new phthalate monoester pollutants in soil and different types of crops is a key technical issue that needs to be urgently addressed. Summary of the Invention

[0006] As described above, the prior art lacks an analytical method for the residual phthalate monoester compounds in samples from crop planting environments. Furthermore, samples from crop planting environments differ from human samples in composition, phthalate monoester compound spectra, and content. This field urgently requires qualitative and quantitative detection of phthalate monoester compounds in samples from crop planting environments, and analysis of phthalate monoester compound spectra in soil and vegetables. The present invention provides a method for detecting residual phthalate compounds in samples from crop planting environments, comprising: (1) extraction: pre-treating samples from the crop planting environment, adding an isotope internal standard, and extracting with an extractant; (2) concentration and enrichment: concentrating the extractant and enriching the target compound through an SPE column; and (3) detection: detecting with HPLC-MS / MS. This study discovered that phthalates are easily converted into phthalate monoesters in agricultural environments. It provides an HPLC-MS / MS analysis method and presents HPLC-MS / MS spectra of 19 typical phthalate monoester compounds, confirming the presence of phthalate monoester residues in soil and five types of vegetables. Compared with direct detection of phthalates, detection of phthalate monoester compounds can more accurately assess the ecological and health risks posed by plasticizers to agricultural cropping systems.

[0007] Specifically, the present invention provides a method for detecting phthalate monoester compound residues in crop planting environment samples, comprising the following steps:

[0008] (1) Extraction: Pre-treat samples from crop planting environments, add isotope internal standards, and extract with extraction solution;

[0009] (2) Concentration and enrichment: The extract is concentrated and passed through a glass SPE column to enrich the target compound;

[0010] (3) Detection: The eluate after SPE enrichment is detected by HPLC-MS / MS, wherein the HPLC-MS / MS is a high performance liquid chromatography coupled with a triple quadrupole mass spectrometer, and the phthalate monoesters are detected in a multiple reaction monitoring mode;

[0011] Wherein, the sample from the crop planting environment is soil and / or vegetables, and the vegetables are selected from peppers, cucumbers, tomatoes, eggplants and lettuce.

[0012] Furthermore, the method for detecting the residues of phthalic acid monoester compounds in crop planting environment samples of the present invention is characterized in that: the phthalic acid monoester compounds are monoethyl phthalate, monoisopropyl phthalate, monobutyl phthalate, mono-n-pentyl phthalate, mono(3-hydroxybutyl) phthalate, 2-hydroxy-isobutyl phthalate, monocyclohexyl phthalate, monohexyl phthalate, mono( phthalate, mono(2-ethyl-5-hydroxyhexyl)phthalate, mono(2-ethyl-5-carboxypentyl)phthalate, mono[2-(hydroxymethyl)ethyl]phthalate, and monocarboxyisooctylphthalate.

[0013] Furthermore, the method for detecting residual phthalate monoester compounds in samples from a crop planting environment of the present invention is characterized in that: the samples from the crop planting environment are pretreated by adding NaCl and Na2SO4 to the samples to avoid the influence of moisture on the extraction effect, preferably 0.2g NaCl and 0.3g Na2SO4 are added to every 2g of sample.

[0014] Furthermore, the method for detecting residual phthalate monoester compounds in crop planting environment samples described in the present invention is characterized in that: an isotope internal standard is added to the pretreated sample before extraction, and the isotope internal standard is selected from monobutyl phthalate-d4, mono-2-ethylhexyl phthalate-d4, and monobenzyl phthalate-d4; preferably, 20 ng of the isotope internal standard compound is added to each gram of sample, and extraction is carried out three times with an acetonitrile solution containing 2% formic acid, and the extracted supernatants are combined.

[0015] Furthermore, the method for detecting residual phthalate monoester compounds in crop planting environment samples described in the present invention is characterized in that the extraction supernatant is blown dry and concentrated with high-purity nitrogen gas, redissolved with ultrapure water, and an HLB glass solid-phase extraction column is selected to enrich and purify the target compound.

[0016] Furthermore, the method for detecting residual phthalate monoester compounds in crop planting environment samples of the present invention is characterized in that the solid phase extraction includes: activating the glass SPE column with methanol and ultrapure water, loading and draining the aqueous solution in the glass SPE column, and eluting the target compound with methanol.

[0017] Furthermore, the method for detecting residual phthalate monoester compounds in crop planting environment samples of the present invention is characterized in that: HPLC uses a C18 chromatographic column, gradient elution, and the initial mobile phase ratio is A:B = 85:15, wherein A: H2O solution containing 10mM acetic acid, B: acetonitrile containing 10mM acetic acid.

[0018] Furthermore, the method for detecting residual phthalate monoester compounds in crop planting environment samples of the present invention is characterized in that the phthalate monoesters are ionized using an electrospray ionization source (ESI) in negative ion mode, and the mass spectrometry conditions are as follows:

[0019]

[0020]

[0021] Furthermore, the method for detecting residual phthalate monoester compounds in crop planting environment samples of the present invention is characterized in that the detection characteristic parameters of the 19 phthalate ester compounds and the 3 isotope internal standards are shown in Table 4.

[0022] Furthermore, the method for detecting residual phthalate monoester compounds in crop planting environment samples of the present invention is characterized by integrating the peak areas of the target compound and the isotope internal standard in the sample, and performing quantitative analysis on the target compound in the crop planting environment sample.

[0023] Furthermore, the method for detecting phthalate monoester residues in crop planting environment samples of the present invention is characterized in that the quantitative analysis adopts a standard curve control method and / or an internal standard method.

[0024] Advantages and beneficial effects of the present invention:

[0025] Sample pretreatment and instrumental analysis methods for vegetable crops are primarily limited to parent compounds of pollutants such as phthalates. Pretreatment and instrumental analysis methods for phthalate monoesters, the endocrine-disrupting metabolites of these pollutants, have not yet been established. This paper establishes a pretreatment and instrumental analysis method for phthalate monoester compounds in complex matrix samples such as soil and different types of crops. Based on this developed method, we have completed the determination and analysis of phthalate monoester compounds in soil and peppers, cucumbers, tomatoes, eggplants, and lettuce, helping to clarify the risk of dietary exposure to these pollutants through vegetables.

[0026] Phthalic acid monoester pollutants usually contain a large number of isomers. The present invention selected 19 typical phthalic acid monoesters, including three pairs of isomers (MHBP and MHiBP, MEHP and MnOP, MECPP and MCMHP). By optimizing the mobile phase composition and gradient elution procedure, the present invention completed the chromatographic separation and precise quantitative analysis of all isomers.

[0027] The present invention uses 2% formic acid in acetonitrile as the extraction solvent. A relatively small volume of extraction solvent is required to effectively extract phthalate monoesters from soil and vegetable samples, with good recovery rates. Compared to the four-step extraction of phthalate monoesters from indoor dust using four different solvent types and ratios, the extraction method developed in this invention is simpler to operate, takes less time, uses fewer organic solvents, and achieves higher extraction efficiency.

[0028] The detection method developed by the present invention is simple, rapid, highly sensitive, reproducible, can effectively separate isomers, and has high qualitative and quantitative accuracy. The developed method can be well applied to the precise quantitative analysis of phthalate monoester compounds in soil and different types of crop samples.

[0029] Specifically:

[0030] First, based on the fact that phthalate compounds are easily degraded into phthalate monoester compounds in crop planting systems and that phthalate monoester compounds are potentially harmful to the human body, the present invention provides a method for assessing plasticizer degradation product pollution by detecting phthalate monoester compounds through SPE-HPLC-MS / MS for common soil and vegetable samples in crop planting systems through sample pretreatment, method selection and parameter optimization.

[0031] Second, the present invention provides SPE-HPLC-MS / MS detection spectra of 19 typical phthalic acid monoester compounds, so that those skilled in the art can determine the spectrum of residual phthalic acid monoester compounds in agricultural environmental samples based on the characteristic peaks of 19 typical phthalic acid monoester compounds according to the method established by the present invention.

[0032] Third, the present invention provides a quantitative standard curve for 19 typical phthalate monoester compounds in agricultural environments, so that the phthalate monoester target compounds in soil and vegetable samples can be quantified by comparison with the standard curve, isotope internal standard method, etc.; and quantitative analysis and detection are achieved in real sample detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without any creative work.

[0034] Figure 1 : Flowchart of sample extraction, instrument analysis and quantitative calculation.

[0035] Figure 2 : XIC chromatograms of 19 phthalic acid monoester compounds and their three isotope-labeled compounds. The compounds corresponding to each peak are as follows:

[0036] 1. MHBP (mono(3-hydroxybutyl)phthalate);

[0037] 2. MCPP (mono(3-carboxypropyl)phthalate);

[0038] 3. MHiBP (2-hydroxyisobutyl phthalate);

[0039] 4. MEP (monoethyl phthalate);

[0040] 5. MiPrP (monoisopropyl phthalate);

[0041] 6. MECPP (mono(2-ethyl-5-carboxypentyl)phthalate);

[0042] 7. MEHHP (mono(2-ethyl-5-hydroxyhexyl)phthalate);

[0043] 8.MBP (monobutyl phthalate);

[0044] 9. MEOHP (mono(2-ethyl-5-oxyhexyl)phthalate);

[0045] 10. MCMHP (mono[2-(hydroxymethyl)ethyl]phthalate);

[0046] 11. MCiOP (monocarboxyethyl octyl phthalate);

[0047] 12.MBzP (monobenzyl phthalate);

[0048] 13. MCHP (monocyclohexyl phthalate);

[0049] 14.MPeP (mono-n-pentyl phthalate);

[0050] 15.MHxP (monohexyl phthalate);

[0051] 16. MiHeP (mono-2-heptyl phthalate);

[0052] 17. MEHP (monoethylhexyl phthalate);

[0053] 18. MnOP (monooctyl phthalate);

[0054] 19.MiNP (monoisononyl phthalate);

[0055] 20.MBP-d4 (monobutyl phthalate-d4);

[0056] 21.Monobenzyl phthalate-d4 (monobenzyl phthalate-d4);

[0057] 22. Monoethylhexyl phthalate-d4 DETAILED DESCRIPTION

[0058] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention. Rather than all embodiments, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. All other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0059] Example 1 Standard curves of 19 phthalate monoester compounds

[0060] The reference materials of 19 phthalate monoester compounds (listed in Table 1) were diluted to 2 mg / L in methanol (HPLC-grade) as stock solutions. These solutions were then serially diluted to create standard solutions with a concentration gradient ranging from 0.001 to 500 μg / L (the isotope concentration in each standard solution was fixed at 40 μg / L). The target compounds in the mixed standard solutions were determined using high-performance liquid chromatography (LC-20AD, SHIMADZU) coupled with a triple quadrupole mass spectrometer (QRAP API 4500, SCIEX). Calibration curves were constructed based on the peak area ratios of the target compounds and their corresponding isotope concentrations. These curves were then used to calculate the concentrations of actual samples, such as soil and vegetables. The limits of detection (LOD) and limits of quantification (LOQ) for these compounds were also evaluated. The calibration curve parameters are shown in Table 2.

[0061] Table 1: Basic information of 19 phthalate monoester compounds

[0062]

[0063]

[0064] Table 2: Quantitative standard curve parameters for 19 phthalate monoester compounds

[0065]

[0066] Example 2 Establishment of sample pretreatment method

[0067] (1) Actual sample cleaning

[0068] Forty-eight 50 mL clear glass centrifuge tubes and other glassware were rinsed three times with tap water, twice with n-hexane (chromatographic grade), and twice with methanol (chromatographic grade). The tubes were then baked in a muffle furnace at 450°C for 4 h to remove impurities. Vegetable samples were pulverized using a stainless steel grinder, placed in aluminum boxes, and stored at -20°C. Two grams of soil and five pulverized vegetable samples (pepper, cucumber, tomato, eggplant, and lettuce) were weighed in quadruplicate (three replicates and one blank), for a total of 24 samples. All samples were weighed twice, for a total of 48 samples, and placed in 48 glass centrifuge tubes. To minimize the effect of moisture in the soil and vegetable samples on the extraction process, 0.2 g of NaCl and 0.3 g of Na₂SO₄ (baked in a muffle furnace at 450°C for 4 h) were added to each glass centrifuge tube. Subsequently, 10 mL of acetonitrile containing 2% formic acid was added to the samples to extract the target compounds. The samples were vortexed at 2500 rpm for 15 minutes using a Kylin-Bell vortex shaker, sonicated for 15 minutes, and centrifuged at 3000 g for 15 minutes. The supernatant was aspirated and placed in an organic waste container. The target compounds in the soil and vegetable samples were then extracted twice using 10 mL of acetonitrile containing 2% formic acid. Repeat the above steps. After the three extractions, the centrifuge tubes were wrapped with aluminum foil, which had been punctured with small holes. The samples were air-dried at room temperature and used for subsequent spike recovery experiments.

[0069] (2) Addition and recovery experiments of mixed standard solutions with different concentrations

[0070] 96 15 mL transparent pointed glass centrifuge tubes and other glassware were rinsed with tap water three times, washed with n-hexane (chromatographic grade) twice, and washed with methanol (chromatographic grade) twice, and then placed in a muffle furnace and baked at 450 °C for 4 h to remove impurities. Air-dried soil and vegetable samples were divided into two groups, each containing four soil samples (three replicates and one blank) and 20 vegetable samples (four samples of each of five vegetable varieties). Both groups were spiked with 10 μL of a 4 mg / L isotope internal standard mixture (butyl phthalate-d4, mono-2-ethylhexyl phthalate-d4, and monobenzyl phthalate-d4). Furthermore, 10 μL of a 4 mg / L and 40 mg / L mixed standard solution containing 19 phthalate monoesters (as shown in Table 2) was added, resulting in an isotope spike concentration of 40 μg / L for both groups. Nonisotopic compounds were spiked at concentrations of 20 μg / L and 200 μg / L, respectively. After standing for 2 hours, the samples were added with 6 mL of acetonitrile containing 2% formic acid to extract the target compounds. The sample was vortexed at 2500 rpm for 15 minutes using a Kylin-Bell vortex shaker, sonicated for 15 minutes, and centrifuged at 3000 g for 15 minutes. The supernatant was aspirated and transferred to a 15 mL glass centrifuge tube. For the remaining two extractions, 5 mL and 4 mL of acetonitrile containing 2% formic acid were added, respectively, and the first extraction procedure was repeated. The supernatants from the three extractions were combined and concentrated to near dryness using high-purity nitrogen. The sample was immediately dissolved in 10 mL of ultrapure water and vortexed at 2500 rpm. An HLB glass solid-phase extraction column (200 mg, 6 mL) was selected to enrich and purify the target compound. a. Activation: 6 mL of methanol and 6 mL of ultrapure water were added in sequence to activate the solid-phase extraction column; b. Loading: 10 mL of the reconstituted sample was loaded at a rate of approximately 1 mL / min using a Supelco 24-tube SPE solid-phase extraction device from the United States. After loading, the vacuum degree was frequently changed using a YM-20 diaphragm vacuum pump to drain the aqueous solution in the solid-phase extraction column; c. Elution: The target compound was eluted from the solid-phase extraction column using 10 mL of methanol solution, and the elution solution was collected using a 15 mL glass centrifuge tube. Finally, the sample was concentrated to near dryness with high-purity nitrogen, immediately dissolved in 1 mL of methanol solution, and vortexed evenly at 2500 rpm using a vortex oscillator. The re-dissolved solution was transferred twice into a 1.5 mL centrifuge tube (with a built-in 0.5 mL glass liner tube). The tube was centrifuged at 10,000 g for 10 minutes using a Thermo high-speed refrigerated centrifuge. The supernatant was immediately transferred to a 1.5 mL brown injection vial and stored in a -20°C refrigerator until tested.

[0071] (3) Parameter optimization of high performance liquid chromatography tandem triple quadrupole mass spectrometer

[0072] The target compounds in the spiked recovered sample extracts were determined using a high performance liquid chromatography (LC-20AD, SHIMADZU) tandem triple quadrupole mass spectrometer (QRAP API 4500, SCIEX). The recovery rate of each target compound was calculated using the formula: recovery rate = (spike sample concentration measurement value - unspike (blank) sample concentration measurement value) / added standard concentration value × 100%. The results of the recovery rates of the two different concentrations of mixed standard solutions in soil and five vegetable samples are shown in Table 3-8.

[0073] Table 3: Recovery rates of soil spiked with different concentrations (20 μg / L and 200 μg / L)

[0074]

[0075] Table 4: Recovery rates of pepper at different spike concentrations (20 μg / L and 200 μg / L)

[0076]

[0077]

[0078] Table 5: Recovery rates of cucumber at different spike concentrations (20 μg / L and 200 μg / L)

[0079]

[0080] Table 6: Recovery rates of different spiked tomato concentrations (20 μg / L and 200 μg / L)

[0081]

[0082]

[0083] Table 7: Recovery rates of eggplant at different spike concentrations (20 μg / L and 200 μg / L)

[0084]

[0085] Table 8: Recovery rates of different spiked concentrations of lettuce (20 μg / L and 200 μg / L)

[0086]

[0087]

[0088] The recoveries of the developed method ranged from 51% to 130% at both spike concentrations in each sample. The precision, expressed as the relative standard deviation (RSD), was 0-18% in soil and vegetable samples. According to the Japanese Ministry of Land, Infrastructure, Transport and Tourism's Manual for the Analysis of Endocrine Disrupting Compounds, recoveries between 50% and 120% with RSDs below 20% are considered acceptable. Previous studies have shown that high recoveries of organic compounds are often due to differences in absolute recoveries between the target compound and its internal standard; therefore, an acceptable recovery range for organic compounds is recommended to be between 50% and 150%. The recoveries of the target compounds in Tables 3-8 demonstrate that the recoveries of all phthalate monoesters studied in this study in soil and five vegetable samples generally met quality control requirements. The linearity, sensitivity, recovery, and precision of this method demonstrate its reliability and suitability for the extraction of similar phthalate monoesters from soil and vegetable samples.

[0089] The purpose of conducting recovery experiments on soil and five vegetable samples with two different concentrations of mixed standard solutions of 20 μg / L and 200 μg / L is to verify that the developed extraction method is reliable and can be used to extract actual samples, ensuring that the target compounds in most actual samples can be effectively extracted by this method.

[0090] The concentration of the internal standard mixed solution added to the actual samples and spiked recovery samples was 40 μg / L after instrumental measurement. At this concentration, the HPLC-MS / MS instrument was most stable and sensitive. Therefore, in the subsequent extraction of actual soil and vegetable samples, a 40 μg / L isotope-labeled internal standard compound mixed solution was added for the quantification of the target phthalate monoester compounds in the actual samples.

[0091] Example 3: Quantitative analysis of phthalate monoester compounds in soil and vegetable samples

[0092] (1) Sample collection and extraction

[0093] Three soil samples and three vegetable samples (pepper, cucumber, tomato, eggplant and lettuce) of five kinds were collected on site, for a total of 18 samples. Twenty 50mL transparent glass centrifuge tubes, 40 15mL transparent pointed glass centrifuge tubes and other glassware were washed with tap water three times, washed with n-hexane (chromatographic grade) twice, and washed with methanol (chromatographic grade) twice, and then placed in a muffle furnace and baked at 450℃ for 4h to remove impurity interference. At the same time, diatomaceous earth was placed in a brown glass bottle and placed in a muffle furnace and baked at 450℃ for 4h to remove impurity interference. For the 18 soil and vegetable samples, 2g of each sample was weighed and placed in a 50mL transparent glass centrifuge tube. In order to minimize the influence of moisture in the soil and vegetable samples on the extraction effect, 2g of diatomaceous earth was added to each glass centrifuge tube.

[0094] 0.2g NaCl and 0.3g Na2SO4 (after baking in a muffle furnace at 450℃ for 4h) were added to the tube. At the same time, 2g diatomaceous earth sample was weighed in duplicate and placed in two 50mL transparent glass centrifuge tubes. 10μL of a 4mg / L isotope internal standard mixed solution (monobutyl phthalate-d4, mono-2-ethylhexyl phthalate-d4, monobenzyl phthalate-d4) was added to each of the 18 actual samples and 2 diatomaceous earth. After standing for 2h, 6mL of acetonitrile solution containing 2% formic acid was added to the sample to extract the target compound. The sample was vortexed at 2500rpm for 15 minutes using a Kylin-Bell vortex shaker, ultrasonicated for 15 minutes, centrifuged at 3000g for 15 minutes, and the supernatant was transferred to a 15mL glass centrifuge tube. For the remaining two extractions, 5 mL and 4 mL of acetonitrile solution containing 2% formic acid were added, respectively, and the first extraction procedure was repeated. The supernatants from the three extractions were combined and purged with high-purity nitrogen until the sample was nearly dry. The sample was immediately dissolved in 10 mL of ultrapure water and vortexed at 2500 rpm. An HLB glass solid-phase extraction column (200 mg, 6 mL) was used to enrich and purify the target compound. a. Activation: 6 mL of methanol and 6 mL of ultrapure water were added sequentially to activate the solid-phase extraction column. b. Sample loading: 10 mL of the reconstituted sample was loaded at a rate of approximately 1 mL / min using a Supelco 24-tube SPE solid-phase extraction apparatus. After loading, the aqueous solution in the solid-phase extraction column was drained using a YM-20 diaphragm vacuum pump with frequent changes in vacuum. c. Elution: 10 mL of methanol was used to elute the target compound from the solid-phase extraction column, and the eluted solution was collected in a 15 mL glass centrifuge tube. Finally, the sample was concentrated to near dryness with high-purity nitrogen, immediately dissolved in 1 mL of methanol solution, and vortexed evenly at 2500 rpm using a vortex oscillator. The re-dissolved solution was transferred twice into a 1.5 mL centrifuge tube (with a built-in 0.5 mL glass liner tube). The tube was centrifuged at 10,000 g for 10 minutes using a Thermo high-speed refrigerated centrifuge. The supernatant was immediately transferred to a 1.5 mL brown injection vial and stored in a -20°C refrigerator until tested.

[0095] (2) Instrumental analysis

[0096] Analytical instrument: High performance liquid chromatography (LC-20AD, SHIMADZU) tandem triple quadrupole mass spectrometer (QTRAP API4500, SCIEX)

[0097] Chromatographic column: Waters BEH C18 column (2.5 μm, 2.1 × 100 mm)

[0098] Column temperature: 40°C

[0099] Mobile phase: A: H2O (containing 10 mM acetic acid), B: acetonitrile (containing 10 mM acetic acid)

[0100] Flow rate: 0.25 mL / min

[0101] Injection volume: 5 μL

[0102] Needle wash solution: methanol: water (v:v = 1:1) mixed solution

[0103] The mobile phase gradient is shown in Table 9:

[0104] Table 9: HPLC gradient elution parameters

[0105]

[0106]

[0107] The phthalic acid monoesters in the mixed standard solution and the sample extract were ionized using the negative ion mode of the electrospray ionization source (ESI) in the mass spectrometer. The mass spectrometry conditions are shown in Table 10:

[0108] Table 10: Mass spectrometry parameters

[0109] parameter name parameter Numerical mass spectrometer SCIEX QTRAP API 4500 Collision gas (CAD) Medium Ion source Electrospray ionization (ESI) Curtain Air (CUR) 35psi Ionization mode Negative ion mode Atomizing gas (Gas1) 50psi Atomizing gas Nitrogen Heating gas (Gas2) 55psi Collision gas Argon Ion spray voltage -4500v Collection method Multiple reaction monitoring (MRM) Temperature (TEM) 450℃

[0110] The multiple reaction monitoring mode (MRM) was used to qualitatively analyze the phthalate monoester compounds. The results are shown in Table 11. Figure 2 As shown:

[0111] Table 11: Characteristic parameters of 19 phthalate monoester compounds and 3 isotopic internal standards

[0112]

[0113]

[0114] (3) Quantitative analysis of target compounds

[0115] Using MultiQuant TM The software (version 3.0, SCIEX) was used to integrate the peak areas of the target compounds and isotopes in the samples, calculate the ratio of the peak area of ​​the target compound to the corresponding isotope, and compare it with the obtained standard curve to quantitatively analyze the target compounds in soil and vegetable samples using the internal standard method.

[0116] (4) Test results

[0117] The quantitative analysis results of the target compounds in soil and five vegetable samples (pepper, cucumber, tomato, eggplant and lettuce) are shown in Table 12. Table 12 shows that of the 19 phthalate monoester compounds involved in this study, 11, 14, 11, 12, 12 and 13 were detected in soil, pepper, cucumber, tomato, eggplant and lettuce samples, respectively. Among them, 6 compounds were detected in both soil and five vegetable samples. The average concentration of phthalate monoesters in soil samples ranged from ND to 6.25 μg / kg, in pepper samples from ND to 33.49 μg / kg, in cucumber samples from ND to 28.37 μg / kg, in tomato samples from ND to 135.52 μg / kg, in eggplant samples from ND to 35.02 μg / kg, and in lettuce samples from ND to 23.5 μg / kg (as shown in the table below). Overall, the concentration of phthalate monoesters in vegetable samples was higher than in soil samples.

[0118] Table 12: Quantitative analysis results of target compounds in soil and five vegetable samples (pepper, cucumber, tomato, eggplant and lettuce)

[0119]

[0120] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting phthalate monoester residues in crop planting environment samples, comprising the following steps: (1) Extraction: Pre-treat samples from crop planting environments, add isotope internal standards, and extract with extraction solution to avoid the influence of moisture on the extraction effect; (2) Concentration and enrichment: The extract was concentrated, reconstituted with ultrapure water, and passed through a glass SPE column to enrich the target compound; (3) Detection: The eluate after SPE enrichment is detected by HPLC-MS / MS liquid chromatography-mass spectrometry, and the HPLC-MS / MS high performance liquid chromatography-tandem triple quadrupole mass spectrometer is used to detect phthalic acid monoesters in multiple reaction monitoring mode; Wherein, the sample from the crop planting environment is soil and / or vegetables, and the vegetables are selected from peppers, cucumbers, tomatoes, eggplants and lettuce; HPLC was performed using a C18 column with gradient elution. The initial mobile phase was A:B = 85:15, where A was H2O solution containing 10 mM acetic acid and B was acetonitrile containing 10 mM acetic acid. The gradient elution mobile phase parameters are as follows: The phthalic acid monoester compound is monoethyl phthalate, monoisopropyl phthalate, monobutyl phthalate, mono-n-pentyl phthalate, mono(3-hydroxybutyl) phthalate, 2-hydroxy-isobutyl phthalate, monocyclohexyl phthalate, monohexyl phthalate, mono(3-carboxypropyl) phthalate, monobenzyl phthalate, mono-2-heptyl phthalate, monoethylhexyl phthalate, monooctyl phthalate, mono(2-ethyl-5-oxyhexyl) phthalate, monoisononyl phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate, mono(2-ethyl-5-carboxypentyl) phthalate, mono[ 2-(carboxymethyl)hexyl] ester and monocarboxyethyl phthalate.

2. The method for detecting phthalate monoester residues in crop planting environment samples according to claim 1, wherein: The pretreatment of samples from crop growing environments was to add NaCl and Na2SO4 to the samples to avoid the influence of moisture on the extraction effect. 0.2g NaCl and 0.3g Na2SO4 were added to every 2g sample.

3. The method for detecting phthalate monoester residues in crop planting environment samples according to claim 1, wherein: Before extraction, isotope internal standards were added to the pretreated samples. The isotope internal standards were monobutyl phthalate-d4, mono-2-ethylhexyl phthalate-d4, and monobenzyl phthalate-d4. 20 ng of the isotope internal standard compound was added to each gram of sample. The sample was extracted three times with an acetonitrile solution containing 2% formic acid, and the extracted supernatants were combined.

4. The method for detecting phthalate monoester residues in crop planting environment samples according to claim 1, wherein: The extraction supernatant was dried and concentrated with high-purity nitrogen, re-dissolved with ultrapure water, and the target compound was enriched and purified using an HLB glass solid phase extraction column.

5. The method for detecting phthalate monoester residues in crop planting environment samples according to claim 1, wherein: The SPE comprises: activating an SPE column with methanol and ultrapure water, loading a sample and draining the aqueous solution in the SPE column, and eluting the target compound with methanol.

6. The method for detecting phthalate monoester residues in crop planting environment samples according to claim 1, wherein: Phthalic acid monoesters were ionized using an electrospray ionization (ESI) source in negative ion mode, and the mass spectrometry conditions were as follows: 。 7. The method for detecting phthalate monoester residues in crop planting environment samples according to any one of claims 1 to 6, characterized in that: The detection characteristic parameters of 19 phthalate compounds and 3 isotope internal standards are shown below. 。 8. The method for detecting phthalate monoester residues in crop planting environment samples according to any one of claims 1 to 6, characterized in that: The peak areas of the target compounds and isotope internal standards in the samples were integrated to quantitatively analyze the target compounds in the crop planting environment samples.

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