A method for determining phthalate esters and their substitutes in tea leaves

Abstract

The application provides a method for determining phthalate esters and substitutes thereof in tea leaves, and relates to the technical field of phthalate ester plasticizer detection, which comprises the following steps: after tea leaves are soaked in water and extracted with an extraction agent, water is removed by using a water removal agent to obtain an extraction liquid; a purifying agent is added to the extraction liquid for purification to obtain a test liquid; and phthalate esters and substitutes thereof in the test liquid are determined by gas chromatography-triple quadrupole mass spectrometry. The application can effectively remove impurities such as pigments, has the advantages of simple operation, convenience, good extraction effect, high sensitivity and good repeatability. Meanwhile, phthalate esters and substitutes thereof in the test liquid are determined by gas chromatography-triple quadrupole mass spectrometry, 20 kinds of phthalate esters and substitutes thereof in tea leaves can be determined at the same time, the sensitivity is high, the time consumption is short, qualitative ions and quantitative ions are verified at the same time, the qualitative and quantitative accuracy is high, and the method is suitable for the detection of various tea leaves.

Description

Technical Field

[0001] This invention relates to the field of phthalate plasticizer detection technology, and in particular to a method for determining phthalates and their substitutes in tea. Background Technology

[0002] Phthalate esters (PAEs) are common plasticizers widely used in various products, including wallpaper, electrical wires, toys, food packaging materials, and blood bags. Due to the various health effects they cause to humans, such as genotoxicity, neurotoxicity, cytotoxicity, reproductive toxicity, and endocrine disruption, they are listed on regulatory lists in many countries. With the increasing regulation of phthalate esters, the production of their alternative plasticizers has been rising year by year, attracting attention. Numerous studies have shown that many PAE substitutes have endocrine disrupting, reproductive toxicity, and developmental toxicity.

[0003] The main routes of exposure to PAEs and their substitutes include dietary intake, inhalation through the air, and skin contact, with dietary intake being the primary source of human exposure. Tea, as the world's second most consumed beverage after water, has a wide consumer base and a high per capita intake. Its growth, harvesting, processing, packaging, and storage can all introduce PAEs and their substitutes, posing a risk of human exposure. Establishing a method for determining multiple types of PAEs and their substitutes in tea can provide a methodological basis for the monitoring and risk assessment of plasticizers in tea. Tea samples have complex matrices and contain a wide variety of PAEs and their substitutes. Highly sensitive instrumental methods and rapid, simple extraction and purification methods are both the focus and the challenge in analysis. However, current research has not focused on the pollution level analysis of PAE substitutes in tea. Existing methods for detecting PAE substitutes in multiple media are mostly column chromatography separation methods, which are cumbersome, time-consuming, prone to introducing contamination, and ineffective at removing pigments from tea. Therefore, there is an urgent need to establish a simple, highly sensitive, and effective trace analysis method for the simultaneous detection of multiple PAEs and their substitutes in tea. Summary of the Invention

[0004] The present invention aims to provide a method for determining phthalates and their substitutes in tea, in order to at least partially solve at least one of the above-mentioned technical problems.

[0005] To address the aforementioned technical problems, the first aspect of this invention provides a method for determining phthalates and their substitutes in tea, comprising:

[0006] The tea leaves are soaked in water and extracted with an extractant. Then, a dehydrating agent is used to remove the water to obtain the extract.

[0007] A purifying agent is added to the extract to purify it, thus obtaining the test solution;

[0008] Phthalate esters and their substitutes in the test solution were determined by gas chromatography-triple quadrupole mass spectrometry.

[0009] In a preferred embodiment, the step of soaking tea leaves in water and extracting them with an extractant, followed by removing moisture with a dehydrating agent to obtain an extract, comprises:

[0010] Add water to tea leaves and steep for a short time to obtain a tea-water mixture;

[0011] Add the extractant to the tea-water mixture, vortex for a second time, add sodium chloride, vortex for a third time, and sonicate for a predetermined time to obtain the extracted mixture;

[0012] Add a dehydrating agent to the extraction mixture, shake and centrifuge, and take the supernatant as the extraction liquid.

[0013] In a preferred embodiment, the first time is 20-40 min, the second time is 25-40 s, the third time is 25-35 s, the predetermined time is 5-20 min, the shaking is 10 min, the centrifugation is 3000 rpm, the volume ratio of water to extractant is 1:2, and the mass ratio of water, sodium chloride and dehydrating agent is 5:1:3.

[0014] In a preferred embodiment, the step of adding a purifying agent to the extract to purify it and obtaining a test solution includes:

[0015] Add a purifying agent to the extract, vortex for a fourth time, shake well, let stand for a fifth time, centrifuge, filter the supernatant to obtain the test solution.

[0016] In a preferred embodiment, the fourth time is 15-40 seconds, the fifth time is 15-30 minutes, the centrifugation is performed at 3500 rpm, and the mass ratio of tea leaves to purifying agent is 1:31.

[0017] In a preferred embodiment, the extractant is a mixture of acetonitrile and toluene.

[0018] In a preferred embodiment, the volume ratio of acetonitrile to toluene in the acetonitrile-toluene mixed solution is 3:1.

[0019] In a preferred embodiment, the dehydrating agent is anhydrous magnesium sulfate.

[0020] In a preferred embodiment, the purifying agent is a mixture of N-propylethylenediamine solid-phase adsorbent, octadecyl silica gel adsorbent, and graphitized carbon black filler.

[0021] In a preferred embodiment, the mass ratio of N-propylethylenediamine solid adsorbent, octadecyl silica gel adsorbent, and graphitized carbon black filler in the purifying agent is 1:1:0.48.

[0022] In summary, the method for determining phthalates and their substitutes in tea according to the present invention involves soaking tea leaves in water and extracting them with an extractant, then removing the water with a dehydrating agent to obtain an extract. A purifying agent is then added to the extract for purification to obtain a test solution. This method effectively removes impurities such as pigments and has the advantages of simple operation, convenience, good extraction effect, high sensitivity, and good repeatability. Furthermore, the method utilizes gas chromatography-triple quadrupole mass spectrometry to determine phthalates and their substitutes in the test solution, enabling the simultaneous determination of 20 phthalates and their substitutes in tea leaves. This method offers high sensitivity, short processing time, dual verification of qualitative and quantitative ions, accurate qualitative and quantitative analysis, and applicability to the detection of various types of tea. Attached Figure Description

[0023] Figure 1 This is a schematic flowchart of a method for determining phthalates and their substitutes in tea according to an embodiment of the present invention;

[0024] Figures 2a-2t The peak spectra (100 μg / L) of standard samples of 20 phthalates and their substitutes are shown. Detailed Implementation

[0025] The features and exemplary embodiments of various aspects of the present invention will now be described in detail. To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only configured to explain the present invention and are not configured to limit the present invention. For those skilled in the art, the present invention can be practiced without some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the present invention by illustrating examples of the invention.

[0026] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes said element.

[0027] This invention provides a method for determining phthalates and their substitutes in tea, wherein: the phthalate compounds include: dimethyl phthalate (DMP), diethyl phthalate (DEP), diisobutyl phthalate (DiBP), dibutyl phthalate (DBP), dihexyl phthalate (DNHP), butyl benzyl phthalate (BzBP), dicyclohexyl phthalate (DCHP), dioctyl phthalate (DnOP), di(2-ethylhexyl) phthalate (DEHP), and didecyl phthalate (DDP); the phthalate substitutes include... Including: dimethyl isophthalate (DMIP), diisobutyl adipate (DiBA), tributyl acetyl citrate (ATBC), di(2-ethylhexyl) adipate (DEHA), di(2-ethylhexyl)cyclohexane-1,2-dicarboxylic acid ester (1,2-DEHCH), bis(2-ethylhexyl) 4-cyclohexenyl-1,2-dicarboxylate (DEHTH), tributyl trimellitate (TBTM), bis(2-ethylhexyl) isophthalate (DEHIP), bis(2-ethylhexyl) terephthalate (DEHTP), and tri(2-ethylhexyl) trimellitate (TOTM). Figure 1 As shown, the method includes:

[0028] S1. After soaking the tea leaves in water and extracting them with an extractant, remove the water with a dehydrating agent to obtain the extract.

[0029] For example, this step may include:

[0030] S11. Add water to the tea leaves and steep for a short time to obtain a tea-water mixture;

[0031] S12. Add the extractant to the tea-water mixture, vortex for a second time, add sodium chloride, vortex for a third time, and sonicate for a predetermined time; to obtain the extracted mixture;

[0032] S13. Add a dehydrating agent to the extraction mixture, shake and centrifuge, and take the supernatant as the extraction liquid.

[0033] Wherein: the first time is 20-40 min, preferably 30 min; the second time is 25-40 s, preferably 30 s; the third time is 25-35 s, preferably 30 s; the predetermined time is 5-20 min, preferably 10 min; shaking for 10 min; and centrifugation at 3000 rpm. Deionized water can be used.

[0034] Preferably, the volume ratio of water to extractant is 1:2.

[0035] Preferably, the mass ratio of water, sodium chloride, and dehydrating agent is 5:1:3.

[0036] In this embodiment, the extractant is preferably an acetonitrile-toluene mixed solution, and the volume ratio of acetonitrile to toluene in the acetonitrile-toluene mixed solution is 3:1.

[0037] The dehydrating agent can be anhydrous magnesium sulfate.

[0038] S2. Add a purifying agent to the extract to purify it and obtain the test solution;

[0039] For example, a purifying agent is added to the extract obtained in step S1, the mixture is vortexed for a fourth time, shaken well, allowed to stand for a fifth time, centrifuged, and the supernatant is filtered through a 0.22μm glass fiber membrane. The filtrate is the test solution.

[0040] The purifying agent is a mixture of N-propylethylenediamine solid-phase adsorbent (PSA), octadecyl silica gel adsorbent (C18), and graphitized carbon black filler (GCB). Preferably, the mass ratio of N-propylethylenediamine solid-phase adsorbent, octadecyl silica gel adsorbent, and graphitized carbon black filler in the purifying agent is 1:1:0.48.

[0041] The fourth time is 15-40s, preferably 30s, and the fifth time is 15-30min, preferably 20min. The centrifugation is carried out at 3500rpm, and the mass ratio of tea leaves to purifying agent is 1:31.

[0042] S3. Phthalate esters and their substitutes in the test solution were determined by gas chromatography-triple quadrupole mass spectrometry.

[0043] For example, gas chromatography-triple quadrupole mass spectrometry (GC-MS / MS) can be used to detect phthalates and their substitutes in the test solution, and the internal standard method can be used for quantification.

[0044] The method for determining phthalates and their substitutes in tea according to the present invention will be described in detail below through specific embodiments.

[0045] A method for determining phthalates and their substitutes in tea includes:

[0046] Step 1: Weigh 0.2g of tea sample and place it in a 10mL glass tube. Add 2mL of water and soak for 30min to obtain a tea-water mixture.

[0047] Step 2: Add 4 mL of acetonitrile-toluene extractant at a volume ratio of 3:1 to the tea-water mixture, vortex for 30 s, add 0.4 g of sodium chloride, vortex for another 30 s, and sonicate for 10 min to obtain the extraction mixture.

[0048] Step 3: Add 1.2g of dehydrating agent to the extraction mixture, shake for 10min, centrifuge at 3000rpm, and take the supernatant as the extraction solution.

[0049] Step 4: Add 0.25g PSA, 0.25g C18, and 0.12g GCB to the extract, vortex for 30s, shake well, let stand for 20min, and then centrifuge at 3500rpm.

[0050] Step 5: Take 1 mL of supernatant and filter it through a 0.22 μm glass fiber membrane. Place the filtrate in a brown vial and wait for GC-MS / MS determination.

[0051] I. GC-MS / MS determination conditions:

[0052] Chromatographic conditions:

[0053] Chromatographic column: TG-5MS column (30m×0.25mm×0.25μm); column temperature: 60℃; injection port temperature: 290℃; carrier gas: He, splitless injection mode, injection volume 2uL; constant flow mode, column flow rate 1.0mL / min; temperature program: initial temperature 60℃, hold for 1min, increase to 220℃ at a rate of 20℃ / min, hold for 1min, then increase to 250℃ at a rate of 5℃ / min, hold for 1min, then increase to 290℃ at a rate of 10℃ / min, hold for 8min; transfer line temperature: 290℃.

[0054] Mass spectrometry conditions:

[0055] Ion source: Electron impact ionization (EI); ionization energy 70 eV, emission current 50 μA; quadrupole temperature 60℃, ion source temperature 290℃; collision gas is high-purity argon (99.999%); ion source temperature: 290℃; multiple reaction monitoring (SRM) mode.

[0056] Other mass spectrometry parameters (parent ion, daughter ion, collision energy, and declustering voltage) of 20 phthalate esters and their substitutes determined in this embodiment are shown in Table 1. Figures 2a-2t The peak spectra of standard samples (100 μg / L) in Table 1 are as follows: DMP, DEP, DiBP, DBP, DNHP, BzBP, DCHP, DnOP, DEHP, DDP, DMIP, DiBA, ATBC, DEHA, 1,2-DEHCH, DEHTH, TBTM, DEHIP, DEHTP, and TOTM.

[0057] Table 1. Mass spectrometric parameters of 20 phthalates and their substitutes

[0058]

[0059]

[0060] II. Linear Range and Method Detection Limit

[0061] Using hexane as a solvent, a mixed standard solution of 20 phthalic acid esters and their substitutes at a concentration of 1 mg / L was serially diluted to prepare a series of mixed standard solutions of 0.01, 0.1, 1, 5, 10, 25, 50, 100 and 200 μg / L. After detection by GC-MS / MS, a standard curve was plotted with plasticizer concentration as the abscissa (x, μg / L) and peak area as the ordinate (y).

[0062] Table 2 shows the detection results of the method limits of detection (LODs), limits of quantitation (LOQs), and linear ranges for 20 phthalate esters and their substitutes. As can be seen from Table 2, the correlation coefficients (r) of the standard curves for all phthalate esters and their substitutes are greater than 0.99. Extrapolating the LODs and LOQs using a signal-to-noise ratio of 3 and 10 times respectively, the LODs and LOQs for the 20 phthalate esters and their substitutes are 0.002–0.634 ng / g and 0.008–2.11 ng / g, respectively.

[0063] Table 2. Limits of detection, limits of quantitation, and linear ranges for 20 phthalates and their alternatives.

[0064] DMP 0.038 0.126 0.01-200 0.997 DMIP 0.050 0.168 0.01-200 0.999 DEP 0.014 0.048 0.01-200 1.000 DiBA 0.030 0.102 0.01-200 0.999 DiBP 0.002 0.008 0.01-200 1.000 DBP 0.048 0.160 0.01-200 0.998 ATBC 0.634 2.11 0.01-200 1.000 DNHP 0.068 0.226 0.01-200 1.000 BzBP 0.034 0.110 0.01-200 1.000 DEHA 0.018 0.064 0.01-200 1.000 1,2-DEHCH 0.020 0.064 0.01-200 1.000 DEHTH 0.042 0.142 0.01-200 1.000 DCHP 0.046 0.154 0.01-200 1.000 DEHP 0.006 0.020 0.01-200 1.000 TBTM 0.022 0.074 0.01-200 1.000 DEHIP 0.160 0.536 0.01-200 1.000 DnOP 0.040 0.136 0.01-200 1.000 DEHTP 0.006 0.018 0.01-200 1.000 DDP 0.546 1.82 0.01-200 1.000 TOTM 0.026 0.086 0.01-200 1.000

[0065] III. Method recovery, limit of detection, and limit of quantitation

[0066] 0.2 g of tea sample was weighed, and phthalic acid esters and their substitutes standards were added to achieve spiked concentrations of 0.25, 0.50, and 1.00 μg / g, with three replicates for each concentration level. The results are shown in Table 3. The recoveries of 20 phthalic acid esters and their substitutes in tea at spiked concentrations of 0.25, 0.50, and 1.00 μg / g ranged from 58.9% to 144%, 64.3% to 121%, and 63.3% to 133%, respectively, with relative standard deviations (RSDs) of 1.69% to 17.6%, 0.72% to 12.2%, and 1.27% to 13.1%, respectively.

[0067] Table 3. Spiked recoveries and relative standard deviations of 20 phthalate esters and their substitutes (n=3)

[0068]

[0069] IV. Determination of actual soil samples

[0070] The method of this invention for determining phthalates and their substitutes in tea was used to analyze 20 phthalates and their substitutes in 6 tea samples. The specific results are shown in Table 4.

[0071] Table 4. Content (ng / g) of 20 phthalates and their substitutes in 6 tea samples

[0072]

[0073]

[0074] Note: nd represents less than the detection limit.

[0075] The above test results show that the method for determining phthalates and their substitutes in tea can effectively remove impurities such as pigments, and has the advantages of simple operation, convenience, good extraction effect, high sensitivity, and good repeatability. Furthermore, by using gas chromatography-triple quadrupole mass spectrometry to determine phthalates and their substitutes in the test solution, 20 kinds of phthalates and their substitutes in tea can be determined simultaneously. This method has high sensitivity, short processing time, and achieves dual verification of qualitative and quantitative ions, ensuring accurate qualitative and quantitative analysis, and is suitable for the detection of various types of tea.

[0076] It should be clarified that the present invention is not limited to the specific structures and processes described above and shown in the figures. For the sake of brevity, detailed descriptions of known methods are omitted here. In the above embodiments, several specific steps are described and shown as examples. However, the method process of the present invention is not limited to the specific steps described and shown. Those skilled in the art can make various changes, modifications, and additions, or change the order of steps, after understanding the spirit of the present invention.

[0077] It should also be noted that the exemplary embodiments mentioned in this invention describe methods or systems based on a series of steps or apparatus. However, this invention is not limited to the order of the steps described above; that is, the steps can be performed in the order mentioned in the embodiments, or in a different order, or several steps can be performed simultaneously.

[0078] The above description is merely a specific embodiment of the present invention. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working processes of the systems, modules, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here. It should be understood that the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the protection scope of the present invention.

Claims

1. A method for determining phthalates and their substitutes in tea, characterized in that, The method includes: Add water to tea leaves and steep for a short time to obtain a tea-water mixture; An extractant consisting of acetonitrile and toluene in a volume ratio of 3:1 was added to the tea-water mixture. After vortexing for a second time, sodium chloride was added, followed by a third vortexing. After sonication for a predetermined time, anhydrous magnesium sulfate was added as a dehydrating agent. The mixture was then shaken and centrifuged, and the supernatant was taken as the extract. The volume ratio of water to extractant was 1:2, and the mass ratio of water, sodium chloride, and dehydrating agent was 5:1:

3. Add a purifying agent to the extract, vortex for four hours, shake well, let stand for five hours, centrifuge, take the supernatant and filter to obtain the test solution; the purifying agent is a mixture of N-propylethylenediamine solid-phase adsorbent, octadecyl silica gel adsorbent and graphitized carbon black filler, with a mass ratio of 1:1:0.

48. Phthalate esters and their substitutes in the test solution were determined by gas chromatography-triple quadrupole mass spectrometry (GC-MS). The chromatographic conditions for GC-MS included: column: TG-5MS column; column temperature: 60℃; injection port temperature: 290℃; carrier gas: He, splitless injection mode, injection volume: 2 μL; constant flow mode, column flow rate: 1.0 mL / min; temperature program: initial temperature 60℃, held for 1 min, increased to 220℃ at a rate of 20℃ / min, held for 1 min, then increased to 250℃ at a rate of 5℃ / min, held for 1 min, then increased to 290℃ at a rate of 10℃ / min, held for 8 min; transfer line temperature: 290℃. The mass spectrometry conditions included: ion source: electron impact ionization source; ionization energy: 70... eV, emission current 50μA; quadrupole temperature 60℃, ion source temperature 290℃; collision gas is 99.999% pure argon; ion source temperature: 290℃; multiple reaction monitoring (SRM) mode; wherein, the phthalate esters include dimethyl phthalate (DMP), diethyl phthalate (DEP), diisobutyl phthalate (DiBP), dibutyl phthalate (DBP), dihexyl phthalate (DNHP), butyl benzyl phthalate (BzBP), dicyclohexyl phthalate (DCHP), dioctyl phthalate (DnOP), di(2-ethylhexyl) phthalate (DEHP), di(2-ethylhexyl) phthalate, ... Decyl ester (DDP); Phthalate substitutes include: dimethyl isophthalate (DMIP), diisobutyl adipate (DiBA), tributyl acetyl citrate (ATBC), di(2-ethylhexyl) adipate (DEHA), di(2-ethylhexyl)cyclohexane-1,2-dicarboxylic acid ester (1,2-DEHCH), bis(2-ethylhexyl) 4-cyclohexenyl-1,2-dicarboxylate (DEHTH), tributyl trimellitate (TBTM), bis(2-ethylhexyl) isophthalate (DEHIP), bis(2-ethylhexyl) terephthalate (DEHTP), and tri(2-ethylhexyl) trimellitate (TOTM).

2. The method according to claim 1, characterized in that, The first time was 20-40 min, the second time was 25-40 s, the third time was 25-35 s, the predetermined time was 5-20 min, the shaking was 10 min, the centrifugation was 3000 rpm, the volume ratio of water to extractant was 1:2, and the mass ratio of water, sodium chloride and dehydrating agent was 5:1:

3.

3. The method according to claim 1, characterized in that, The fourth time was 15-40 seconds, and the fifth time was 15-30 minutes. The mixture was centrifuged at 3500 rpm, and the mass ratio of tea leaves to purifying agent was 1:31.

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