Method for quantitatively analyzing migration content of bisphenol and alkylphenol in polypropylene material by using internal standard method

Abstract

The invention relates to the technical field of quantitative analysis by an internal standard method, in particular to a method for quantitatively analyzing the migration content of bisphenol and alkylphenol in a polypropylene material by an internal standard method, which comprises the following steps: setting migration parameters of 4% acetic acid-water simulation liquid, 10% ethanol-water simulation liquid and olive oil simulation liquid; setting solid-phase extraction purification parameters of the migration liquid; setting concentration and redissolution parameters of the purified liquid; and setting analysis quantitative parameters of the liquid chromatograph-mass spectrometer of the to-be-detected liquid. According to the method, the interference in the matrix is separated by utilizing a specific retention mechanism of solid-phase extraction; according to the present invention, with the high selectivity ion channel monitoring function of the liquid chromatography-mass spectrometer, the accurate identification of the specific component in the complex matrix is achieved based on the mass-to-charge ratio difference of the target object characteristic ion pair, the accurate quantification of the bisphenol and alkylphenol migration trace component is ensured when the isotope internal standard method is combined for quantification, and the method has characteristics of simple pre-treatment operation, high sensitivity and high sensitivity. The method has the advantages of high qualitative analysis accuracy and good trace determination reproducibility.

Description

Technical Field

[0001] The invention relates to the technical field of internal standard method quantitative analysis, in particular to a method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials by using the internal standard method. Background Art

[0002] Polypropylene is a thermoplastic formed from the polymerization of propylene monomers. It exhibits excellent chemical stability, heat resistance, and mechanical strength, making it widely used in food packaging, medical devices, automotive parts, and other fields. During its production process, additives such as antioxidants, plasticizers, and stabilizers are often added to improve processing properties. Some of these additives may contain environmental endocrine disruptors (EDCs). EDCs include bisphenols and alkylphenols. Long-term exposure to bisphenols can cause reproductive system abnormalities, developmental abnormalities, obesity, and even cancer. Alkylphenols can activate estrogen receptors, interfere with natural hormone levels, and affect metabolism and development. Long-term exposure can weaken immune function.

[0003] Currently, the detection of environmental endocrine disruptors primarily relies on chromatography-mass spectrometry (GC-MS). Detection methods for bisphenols and alkylphenols primarily include high-performance liquid chromatography-ultraviolet detection, fluorescence detection, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) has become a mainstream detection method due to its high sensitivity, strong selectivity, and excellent structural elucidation capabilities. However, the high cost of instrumentation, high operating and maintenance costs, and complex operation limit its widespread application in primary laboratories or routine testing. Therefore, there is an urgent need to develop a detection method for bisphenols and alkylphenols in polypropylene materials that can reduce equipment dependence and operational difficulty while ensuring detection accuracy and sensitivity, thereby meeting the needs of daily supervision and quality control. Summary of the Invention

[0004] In response to the above-mentioned technical problems of the high price and high usage threshold of the ultra-high performance liquid chromatography-tandem mass spectrometer used in the existing ultra-high performance liquid chromatography-tandem mass spectrometry method, the technical solution adopted by the present invention to solve the technical problem is: A method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials using an internal standard method comprises the following steps: Step S101, "Sample Pretreatment": Cut the polypropylene material into sample blocks, add an internal standard solution to a simulated immersion solution to perform a migration test, and use a 4% by volume aqueous acetic acid solution, a 10% by volume aqueous ethanol solution, or olive oil as the simulated immersion solution to perform the migration test to obtain a solution to be purified; Specifically, the present invention introduces an internal standard with similar properties to the target compound to correct for systematic errors such as instrument fluctuations and injection volume differences. Alternatively, in some embodiments, such as in liquid chromatography-tandem mass spectrometry, the internal standard and the target compound undergo the same chromatographic retention and mass spectrometric ionization processes, and their response ratio can effectively offset matrix effects and ensure the accuracy of migration calculations.

[0005] Specifically, a 4% aqueous acetic acid solution (simulating acidic foods), a 10% aqueous ethanol solution (simulating alcoholic foods), and an olive oil solution (simulating lipid foods) are used as simulated immersion solutions. These solutions are suitable for full-scale migration analysis of tableware, packaging materials, and other applications. The detection method of the present invention is compatible with the simultaneous analysis of bisphenols and alkylphenols, avoiding repeated testing and improving detection efficiency. By directly detecting the target, derivatization treatments such as silanization reactions required by fluorescence detection or gas chromatography-mass spectrometry techniques are avoided, thereby shortening pre-processing time.

[0006] Step S102, solid phase extraction purification: the liquid to be purified is transferred to a solid phase extraction column, washed with pure water and drained, the target compound is eluted with a methanol-dichloromethane mixed solvent, and the eluate is collected; The present invention uses an HLB or C18 solid-phase extraction column to purify the migration fluid, removing interfering substances such as plasticizers and antioxidants present in the polypropylene material and reducing background noise. Water-soluble impurities are removed by washing with pure water, and the target substance is eluted with a methanol-dichloromethane mixed solvent, balancing the hydrophilicity of bisphenols and the hydrophobicity of alkylphenols, thereby improving the recovery efficiency of the target substance. By reducing matrix interference with liquid chromatography-tandem mass spectrometry detection, the present invention improves the signal-to-noise ratio, further enhancing the intensity and accuracy of the target substance signal during subsequent detection, facilitating more accurate determination of its content.

[0007] Step S103, sample volume adjustment and filtration: the eluate is concentrated to near dryness by nitrogen purge, the volume is adjusted to 1 mL with methanol-5 mmol / L ammonium acetate solution, and filtered through a nylon microporous filter membrane; Specifically, filtration through a 0.22μm nylon microporous filter membrane can remove particulate matter, prevent clogging of the liquid chromatography-tandem mass spectrometry system chromatographic column and contamination of the mass spectrometry ion source, and extend the instrument maintenance cycle. The concentration operation can increase the concentration of bisphenols and alkylphenols targets in the solution, which may originally be at a lower concentration level, making it easier for subsequent liquid chromatography-tandem mass spectrometry to detect these targets more sensitively, especially for trace migration substances with low content, which can also be effectively detected, thereby enhancing the detection ability of the entire detection method for low-content targets. After nitrogen blowing and concentration to near dryness, the volume is adjusted to 1mL with methanol-5mmol / L ammonium acetate solution, which simplifies the re-dissolution step. At the same time, the ammonium acetate buffer can maintain the ionization efficiency of the target and improve the stability of the mass spectrometry response.

[0008] Step S104, instrumental analysis and quantification: liquid chromatography-tandem mass spectrometry is used for detection, and the migration amount of the target substance is calculated by the internal standard method based on pre-established standard curves for bisphenols and alkylphenols.

[0009] Specifically, the present invention is based on a conventional liquid chromatography-tandem mass spectrometry platform, eliminating the need for ultra-high performance liquid chromatography (ULHPLC) systems and reducing laboratory equipment investment costs. Furthermore, conventional liquid chromatography-tandem mass spectrometry platforms require lower-quality instrumentation and sample pretreatment than ULHPLC systems. The present invention eliminates matrix interference through internal standard calibration and solid-phase extraction cleanup, making it particularly suitable for corporate R&D, third-party testing, and quality control by regulatory agencies.

[0010] Furthermore, the environmental endocrine disrupting substances measured in the polypropylene material include bisphenol A, bisphenol B, bisphenol F, tetrachlorobisphenol A, octylphenol, 4-t-octylphenol, nonylphenol, and 4-nonylphenol.

[0011] Specifically, bisphenol A, bisphenol B, bisphenol F, and tetrachlorobisphenol A can cover common bisphenol derivatives and halogenated bisphenols, and octylphenol, 4-t-octylphenol, nonylphenol, and 4-nonylphenol can cover C8-C9 alkyl chain isomers.

[0012] Alternatively, in some embodiments, a gradient elution procedure can be used to achieve baseline separation of hydrophilic bisphenols and hydrophobic alkylphenols having large polarity differences, thereby avoiding co-elution interference.

[0013] Alternatively, in some embodiments, HLB solid-phase extraction columns can be used to efficiently remove interference from oils, organic acids, and other substances in polypropylene migration buffers, achieving a matrix effect suppression rate of ≤20%. A methanol-dichloromethane mixed eluent provides balanced elution efficiency for both polar bisphenols and non-polar alkylphenols, with a target recovery rate of ≥90%. Using a conventional C18 column reduces consumables costs and is suitable for routine testing in small and medium-sized laboratories.

[0014] Specifically, the internal standard method can correct systematic errors such as instrument fluctuations and injection volume differences by introducing an internal standard with similar properties to the target substance, ensuring the accuracy of migration calculation. In particular, for bisphenols and alkylphenols with similar structures and difficult to separate, the internal standard method can effectively improve the reliability of quantitative results. This method can simultaneously detect a variety of bisphenols and alkylphenols, covering common environmental endocrine disruptors. It has the advantages of broad-spectrum coverage of multiple targets, high-sensitivity detection, anti-matrix interference purification and low-cost operation. It is suitable for the detection of polypropylene materials from different sources and uses. Whether it is food contact materials, medical devices or children's toys, this method can be used for risk assessment.

[0015] Furthermore, the internal standard solution in step S101 is an isotope-labeled bisphenol and / or alkylphenol compound, including bisphenol A-d4, bisphenol Bd 10 , bischlorobisphenol A- 13 C 12 , octylphenol-d 17 ,4-t-octylphenol- 13 C6, nonylphenol-d5.

[0016] Specifically, bisphenol A-d4, bisphenol Bd 10 , Tetrachlorobisphenol A-¹³C 12 Corresponding to different bisphenol derivatives, to avoid signal interference between homologues, octylphenol-d 17 4-t-Octylphenol-¹³C6 and nonylphenol-d5 match C8-C9 alkylphenol isomers, resolving the challenge of distinguishing structural analogs by mass spectrometry. 4-t-Octylphenol-¹³C6 and nonylphenol-d5 can clearly distinguish octylphenol / nonylphenol isomers, such as linear and branched alkyl groups, avoiding the risk of misidentification.

[0017] Additionally, the isotope internal standard has the same polarity, retention time, and extraction efficiency as the target, and signal differentiation is achieved through differentiated mass numbers in the mass spectrum. Optionally, in some embodiments, the response ratio of the target and the internal standard is monitored synchronously, and the loss and matrix effect in pre-treatment such as solid phase extraction and nitrogen concentration can be accurately corrected. The recovery fluctuation range is reduced from 50%-150% when there is no internal standard to 85%-115%. The mass spectrometric signal of the isotope internal standard is completely separated from the target, and the interference of the natural isotope peak can be avoided. The detection limit is further reduced to 0.005-0.02 μg / kg. In liquid chromatography-tandem mass spectrometry analysis, injection volume deviation, ion source stability, or chromatographic column performance fluctuations may cause changes in the target signal, while the response changes of the isotope internal standard are synchronized with the target. These systematic errors can be reduced by internal standard correction, and the repeatability and accuracy of the quantitative results can be improved.

[0018] Specifically, while traditional external standard methods require frequent calibration of the standard curve, internal standard methods achieve quantification through a single-point correction, making them particularly suitable for batch sample analysis. Alternatively, in some embodiments, internal standard methods can save calibration time compared to external standard methods. For complex matrices, such as olive oil simulant, multiple matrix-matched standard curves must be prepared. Using isotopic internal standards, a single standard curve is required to cover different matrices, streamlining the method development process.

[0019] Alternatively, in some embodiments, the high sensitivity and stability of isotopic internal standards can reduce the number of repeated analyses, and long-term use can reduce overall testing costs. The accuracy of internal standard methods can reduce the amount of sample or solvent required to improve the signal-to-noise ratio. Alternatively, in some embodiments, using an internal standard method can reduce the amount of methanol-dichloromethane mixed solvent used in the solid-phase extraction elution step by 20% compared to using an external standard method.

[0020] Furthermore, the extraction process of the olive oil test in step S101 includes adding 10 mL of n-hexane and vortex mixing, and then adding 10 mL of methanol-water solution for back extraction to transfer the target product to the lower layer of methanol-water solution.

[0021] Specifically, the distribution behavior of bisphenols and alkylphenols in the n-hexane-methanol-water system is different from that of impurities such as lipids and pigments, and efficient purification can be achieved through two-phase distribution. Olive oil is a high-lipid matrix, and the target bisphenols and alkylphenols have a low distribution coefficient in lipids. However, n-hexane has strong solubility for lipids and can fully dissolve lipid components such as triglycerides in olive oil to form an upper oil phase. After vortex mixing, the lipids are completely extracted into the n-hexane layer, while the target remains in the aqueous phase or interface layer, achieving preliminary separation. Compared with traditional lipid matrix extraction requiring multiple liquid-liquid extractions or solid-phase extraction cleanups, this method can shorten the operation time. The methanol-water extract can be directly used for solid-phase extraction column purification without the need for additional concentration or redissolution steps, avoiding target loss. This method can improve the target recovery rate and detection sensitivity by completely removing the lipid matrix; it also prevents lipids from contaminating the chromatographic column and ion source in subsequent liquid chromatography-tandem mass spectrometry analysis, extending the instrument maintenance cycle.

[0022] Additionally, in some embodiments, lipids can easily cause ion suppression in liquid chromatography-tandem mass spectrometry, reducing the signal intensity of the target compound. Removing lipids through n-hexane extraction reduces matrix interference in the methanol-water extract by over 90%, significantly improving the signal-to-noise ratio.

[0023] Optionally, in some embodiments, the volume ratio of methanol to water is between 6:4 and 8:2. Bisphenols and short-chain alkylphenols have moderate polarity and are more soluble in the methanol-water phase, resulting in stripping recoveries of 85%-95%. Long-chain alkylphenols, such as nonylphenol, are highly hydrophobic. When the volume ratio of methanol to water is 7:3, a recovery rate exceeding 80% can be achieved. Optionally, in some embodiments, when detecting nonylphenol, the mass spectrometer response intensity is increased by 3 times after lipid removal, reducing the limit of detection (LOD) from 5 ng / g to 1.5 ng / g. Furthermore, in some embodiments, the stripping step requires only 10 mL of solvent, compared to the 30-50 mL required for solid-phase extraction cleanup. This method can reduce waste liquid treatment costs. Compared to traditional gas chromatography-mass spectrometry techniques that require oil derivatization, the present invention can simplify the process and reduce the use of toxic reagents.

[0024] Furthermore, the solid phase extraction column in step S102 is a polystyrene divinylbenzene pyrrolidone solid phase extraction column or a solid phase extraction column of model HLB-M-50, and the activation condition of the solid phase extraction column is to sequentially pass 6 mL of methanol and pure water through the column at a flow rate of 1-2 mL / min.

[0025] Specifically, the polystyrene-divinylphenyl matrix forms a porous structure with a high degree of cross-linking through copolymerization, and has a strong hydrophobic retention capacity for bisphenols and alkylphenols. Compared with the C18 column, the polystyrene-divinylphenyl column has a higher recovery rate for weakly polar targets. For example, in some embodiments, the recovery rate of bisphenol A is increased from 85% to 95%. At the same time, the retention of polar impurities such as sugars and organic acids is weaker, and the purification effect is better. The amide group in the pyrrolidone functional group gives the column a medium polarity, which can form hydrogen bonds or dipole-dipole interactions with the target, thereby enhancing the retention of weakly polar substances. In the pure water activation stage, the NVP group of N-vinyl pyrrolidone balances the polarity of the column through hydration, avoids the loss of the target during the activation process, and reduces the nonspecific adsorption of polar impurities such as salts.

[0026] Specifically, 6 mL of methanol fully soaks the styrene-divinylbenzene hydrophobic groups of the HLB packing, opening the pore structure and eliminating air bubble interference, thus preventing a decrease in adsorption capacity due to insufficient activation. Methanol also dissolves and elutes residual oligomers or additives from the column manufacturing process, preventing interference with target detection. Furthermore, passing 6 mL of methanol at a flow rate of 1-2 mL / min ensures adequate matrix wetting without excessive column pressure due to excessive flow rates.

[0027] Additionally, pure water fully hydrates the NVP groups of N-vinyl pyrrolidone, adjusting the polarity of the column surface to a neutral environment, adapting to the subsequent loading of aqueous samples and preventing premature penetration of the target due to sudden changes in solvent polarity. The pyrrolidone groups can form moderate interactions with the target molecules through hydrogen bonds, π-π interactions, etc., allowing bisphenols and alkylphenols to be effectively adsorbed on the column when passing through the extraction column, while some impurities in the sample that have significant differences in properties from the target are not easily adsorbed. The weak interaction between the pyrrolidone group and the target prevents elution difficulties caused by strong retention, ensuring that the target is completely recovered in the subsequent elution step. The hydrated column significantly reduces the retention of polar impurities such as proteins and polysaccharides, which can improve the selectivity of the target. Additionally, a flow rate of 1-2 mL / min ensures that water molecules fully interact with the NVP groups of N-vinyl pyrrolidone, avoiding extended analysis times due to excessively slow flow rates. The activated polystyrene divinylbenzene pyrrolidone solid phase extraction column is compatible with extraction solutions in various solvent systems such as methanol-water and acetonitrile-water, without the need for additional method adjustments.

[0028] Optionally, in some embodiments, the HLB-M-50 solid-phase extraction column is selected from Biocomma and contains a specific ratio of hydrophilic and hydrophobic groups: the hydrophobic divinylbenzene structure retains non-polar compounds, while the hydrophilic N-vinylpyrrolidone structure retains polar compounds. This filler has excellent water wettability and provides good recovery rates for non-polar to moderately polar acidic, neutral, and basic compounds. Biocomma's HLB solid-phase extraction column has a high specific surface area, strong adsorption capacity, high analyte retention, low breakthrough, and low elution volume. Its adsorption capacity and sample capacity are 3-10 times higher than those of C18 bonded silica gel. Biocomma's HLB solid-phase extraction column has an adsorbent specific surface area of 600 m² / g, a uniform particle size of 40 μm, and an average pore size of 300 Å.

[0029] Optionally, in some embodiments, the HLB column can effectively adsorb trace amounts of residual triglycerides in the methanol-water phase after stripping of the olive oil simulated solution, preventing them from entering the mass spectrometry system and contaminating the ion source. The HLB filler used in the present invention is stable within the pH range of 1-12. The pyrrolidone group exhibits high chemical stability, exhibiting no significant degradation within the pH range of 2-8. It is compatible with both 4% acetic acid and 10% ethanol simulated solutions, avoiding the hydrolysis of fillers that occurs in traditional silica-based C18 columns due to acidic conditions. The purified extract reduces the content of impurities such as lipids and pigments by over 95%, minimizing ion suppression in liquid chromatography-tandem mass spectrometry analysis.

[0030] Specifically, the fixed dosage of 6 mL each of methanol and pure water, along with a flow rate range of 1-2 mL / min, facilitates standardized execution by laboratory operators and reduces human error. In some embodiments, compared to the multi-step activation process involving acid-base pretreatment, the present method simplifies the process, shortening the single activation time to less than 10 minutes.

[0031] Alternatively, in some embodiments, the method for preparing polystyrene divinylbenzene pyrrolidone as an adsorbent comprises the following steps: T1. Dissolve methacrylic acid and fatty alcohol polyoxyethylene-polyoxypropylene ether in an aqueous solution containing 20% ethanol, add polyvinyl pyrrolidone and NaCl in sequence, and stir evenly. Add styrene in a semi-continuous addition manner and stir to form an emulsion. Dissolve potassium persulfate in water at a dropping rate of 1 mL / min. Heat to 95°C under nitrogen protection, stir for 2 hours, wash by centrifugation, and dry to obtain polystyrene microspheres. T2. Disperse polystyrene microspheres in a 1% polyvinyl alcohol solution, sonicate for 30 minutes, mix divinylbenzene and a porogen (toluene and n-heptane in a volume ratio of 1:2), slowly add the mixture dropwise to the microsphere dispersion, stir at room temperature for 12 hours to allow swelling, heat to 70°C, add azobisisobutyronitrile to initiate a crosslinking reaction, continue for 8 hours, cool, filter, and extract with acetone Soxhlet for 24 hours to remove the porogen, and vacuum dry to obtain crosslinked polystyrene microspheres; T3. Disperse cross-linked polystyrene microspheres in a 3:1 ethanol / water mixture by volume. Add N-vinyl pyrrolidone and benzoyl peroxide. Purge with nitrogen for 30 minutes and react at 65°C for 12 hours. Filter, wash with methanol to remove unreacted NVP, and vacuum dry to obtain polystyrene divinylbenzene pyrrolidone.

[0032] Specifically, the amount of fatty alcohol polyoxyethylene-polyoxypropylene ether added is 1-3% of the mass of styrene. The fatty alcohol polyoxyethylene-polyoxypropylene ether uses CPE-1500, which has strong emulsification and low foaming properties. It is not easy to generate bubbles during stirring, which helps to form a stable emulsion. The amount of methacrylic acid added is 5-10% of the mass of styrene. Adding a small amount of methacrylic acid can introduce carboxyl functional groups, improve the surface polarity and dispersion stability of the microspheres, and provide reaction sites for subsequent grafting. The amount of NaCl used is 1-3% of the mass of ethylene. NaCl can adjust the ionic strength and control the particle size distribution at 500-800nm. The amount of polyvinyl pyrrolidone added is 1-3% of the mass of styrene, which helps to stabilize the emulsion system and disperse in the subsequent microsphere molding process. Potassium persulfate is used as an initiator, which is 0.5%-1% of the total mass of the monomer to initiate the polymerization reaction.

[0033] Specifically, a 1% polyvinyl alcohol solution is used as a dispersion medium to evenly disperse the polystyrene microspheres, ensuring uniformity in subsequent reactions. Divinylbenzene is used as a crosslinker to form a cross-linked structure in the polystyrene microspheres, improving their mechanical strength and stability. A porogen (toluene and n-heptane, in a 1:2 volume ratio) is used to form a mesoporous structure during the crosslinking process, facilitating the subsequent adsorption of the target compound. Azobisisobutyronitrile is used as an initiator to initiate the crosslinking reaction, with an addition level of 0.2-0.5% of the polystyrene microspheres. Acetone is then used to remove the porogen during Soxhlet extraction.

[0034] Specifically, polystyrene microspheres were dispersed in a 1% polyvinyl alcohol solution and sonicated for 30 minutes to uniformly disperse the microspheres in the solution and prevent agglomeration. The mixed divinylbenzene and porogen were then slowly added dropwise to the microsphere dispersion with gentle stirring. After the addition was complete, stirring was continued at room temperature for 12 hours to allow the divinylbenzene to fully swell within the microspheres and prepare for the crosslinking reaction. After swelling was complete, the reaction system was heated to 70°C, and azobisisobutyronitrile was added to initiate the crosslinking reaction. Stirring was continued for 8 hours, maintaining a stable reaction temperature to ensure full crosslinking and a stable crosslinked microsphere structure. After the reaction was completed, the reaction solution was cooled to room temperature, filtered through filter paper, and the crosslinked microspheres were collected. The crosslinked microspheres were then transferred to a Soxhlet extractor using acetone as the extraction solvent and subjected to Soxhlet extraction for 24 hours to thoroughly remove the porogen and impurities such as residual unreacted starting materials. After the Soxhlet extraction, the microspheres were removed and vacuum dried in a vacuum oven at 50-60°C to a constant weight, yielding crosslinked polystyrene microspheres.

[0035] Specifically, a porogen mixture of toluene and n-heptane forms a mesoporous structure. Soxhlet extraction completely removes the porogen, preserving the porous structure. This results in porous microspheres with a controllable degree of crosslinking, high mechanical strength, and resistance to organic solvents. By adjusting the porogen ratio, the micropore size can be continuously adjusted from micropores (<2 nm) to mesopores (2-50 nm), adapting to the adsorption requirements of target molecules of varying sizes. An ethanol / water mixture balances microsphere dispersibility with N-vinylpyrrolidone solubility. The targeted introduction of a polyvinylpyrrolidone layer onto the microsphere surface imparts a hydrophilic-lipophilic balance, enhancing the adsorption capacity for polar compounds. Benzoyl peroxide initiates the graft polymerization of N-vinylpyrrolidone onto the microsphere surface, forming a hydrophilic polyvinylpyrrolidone layer. The carboxyl groups introduced in step T1 and the grafted polyvinylpyrrolidone in step T3 act synergistically, conferring both polar and nonpolar sites on the microsphere surface, enabling efficient enrichment of pollutants such as bisphenol A and nonylphenol. The hydrophilic polyvinylpyrrolidone layer enhances its affinity for polar compounds, while the hydrophobic polystyrene-divinylbenzene core retains its adsorption capacity for non-polar substances, enabling the simultaneous extraction of multiple contaminants from complex samples. Specifically, the polyvinylpyrrolidone layer exhibits "reverse-phase adsorption" properties, which repel polar matrices such as proteins and carbohydrates, reducing nonspecific adsorption and improving extraction selectivity. Methanol washing effectively removes homopolymers, ensuring grafting efficiency.

[0036] Specifically, the mass ratio of styrene to divinylbenzene is 8:2-6:4, the mass ratio of N-vinylpyrrolidone to cross-linked polystyrene microspheres is 1:5-1:10, and the amount of benzoyl peroxide added is 0.05-0.1 of the mass of the cross-linked polystyrene microspheres. The resulting HLB solid-phase extraction column has an adsorbent specific surface area of 850 m² / g, a uniform particle size of 60 μm, and an average pore size of 300 Å.

[0037] In addition, the injection volume of liquid chromatography-tandem mass spectrometry is in the range of 10-50μL, and can be adapted to solid-phase extraction columns with a variety of different particle size fillers. The conventional filler particle size range is relatively wide, and adsorbents with a particle size of 40-60μm can be used. The injection volume of ultra-high performance liquid chromatography-tandem mass spectrometry is in the range of 1-10μL. In order to better match the ultra-high performance liquid chromatography column, the filler particle size of the solid-phase extraction column is about 20-40μm, thereby achieving more refined separation and enrichment effects in the sample pretreatment stage, reducing the problem of loose front-to-back connection due to differences in filler particle size, affecting the overall detection efficiency and accuracy.

[0038] Furthermore, the liquid chromatography conditions in step S104 are: C18 chromatographic column, mobile phase of methanol and 5 mmol / L ammonium acetate solution gradient elution.

[0039] Specifically, the octadecylsilane bonded phase of a C18 column retains target compounds through hydrophobic interactions. Bisphenols and alkylphenols are strongly adsorbed due to the hydrophobicity of the benzene rings and alkyl chains. Compared to C8 or phenyl columns, C18 columns offer higher resolution (Rs ≥ 1.5) for homologues with smaller polarity differences, such as bisphenol A and bisphenol F, thus preventing coelution from interfering with quantification. Compared to sub-2μm UPLC columns, C18 columns are more tolerant of particulate matter in the sample, reducing the risk of column clogging.

[0040] Specifically, gradient elution dynamically adjusts the elution intensity, eluting highly polar bisphenols in the low organic phase and hydrophobic alkylphenols in the high organic phase, with the peaks eluting sequentially to avoid co-elution interference. An initial low organic phase, such as 10% methanol, can enhance the flushing of polar impurities such as organic acids and sugars in the polypropylene migration buffer, reducing the entry of matrix interferences into the mass spectrometry system. By gradually increasing the methanol ratio, the elution window of the target is precisely controlled, preventing highly hydrophobic interferences such as the antioxidant BHT from co-eluting with the target.

[0041] Specifically, 5mmol / L ammonium acetate solution provides a weakly acidic environment, which can inhibit the dissociation of the target in the mass spectrometry ionization source, reduce the ion suppression effect, promote the deprotonation of bisphenols and alkylphenols in the negative ion mode of electrospray ionization, and improve the ionization efficiency. The ammonium ions in the buffer can compete for the adsorption of silanol groups on the chromatographic column, reduce the secondary interaction between the target and the column, and thus improve the peak shape. Ammonium acetate is a volatile buffer salt that can avoid the crystallization of traditional phosphates or borates in the mass spectrometry and block the ion source, reducing the frequency of instrument maintenance. Furthermore, methanol has a stronger protonation ability than acetonitrile, and synergistically optimizes the ionization efficiency of the target with ammonium acetate, especially for the mass spectrometry response of alkylphenols. In addition, the ammonium acetate buffer in the gradient elution can neutralize the free fatty acids in the sample, avoid the hydrolysis of the silica matrix caused by the strong acid / strong base mobile phase, and extend the column life.

[0042] Furthermore, the method for establishing a standard curve includes: preparing a standard solution containing gradient concentrations of bisphenols, alkylphenol compounds and a fixed concentration of internal standard, performing linear fitting with the peak area ratio of the target substance to the internal standard as the vertical axis and the concentration as the horizontal axis to obtain a standard curve, substituting the measured peak area ratio of the target substance to the internal standard into the standard curve equation to achieve accurate quantification of the analyte.

[0043] The method of the present invention utilizes internal standard correction, wide dynamic range gradient design, and simultaneous multi-target fitting to enable quantitative analysis of bisphenols and alkylphenols in polypropylene migration buffers. The internal standard method corrects systematic errors through ratios, allowing for single-point calibration under stable instrument conditions without the need for multi-point calibration curves. This eliminates the drawback that absolute response values are affected by instrument status, saving standard material consumption and calibration time. While traditional methods require the preparation of matrix-matched standard curves for different matrices, such as olive oil and water-based simulants, the internal standard method corrects for matrix effects through ratios, enabling quantification using only a solvent standard curve. This method is suitable for rapid screening of multi-matrix samples.

[0044] Furthermore, in step S101, in the acetic acid and ethanol aqueous solution tests, 50 mL of the simulated liquid and the internal standard solution were added, and the mixture was thermostated at 100° C. for 30 minutes and then cooled; in the olive oil test, 50 g of olive oil and the internal standard solution were added, and after thermostating, 10 g of the oil sample was mixed with n-hexane and methanol-water solution, and the lower layer solution was collected after centrifugal stratification, concentrated to less than 3 mL by nitrogen blowing, and cooled for purification.

[0045] Specifically, at a constant temperature of 100°C, the migration rate of target substances such as bisphenol A and nonylphenol from the material in the acetic acid and ethanol aqueous solution simulant was significantly accelerated. Simultaneously, high temperature enhanced the solubility of the target substances in the simulant. Compared with room temperature treatment, 30 minutes of high temperature can increase the migration of the target substances by 2-3 times, shortening the migration time and improving detection efficiency. Directly adding a fixed concentration of internal standard solution to the simulant ensures that the target substance and the internal standard experience exactly the same conditions during the migration process, avoiding concentration fluctuations caused by step-by-step addition in subsequent operations. This improves the consistency of the recovery rates of the internal standard and the target substance (RSD <5%), makes quantitative results more accurate, eliminates the need for additional correction of the internal standard addition volume, and reduces human error. Immediate cooling after high-temperature treatment, such as in an ice water bath, can quickly terminate the further migration of the target substance from the material, avoid back adsorption or degradation of the migrating substance due to temperature drop, and ensure the stability of the test results.

[0046] Specifically, 10g of olive oil is mixed with n-hexane to form a homogeneous oil solution, fully releasing bisphenol A and nonylphenol adsorbed in the oil. After adding a methanol-water solution, the mixture is centrifuged (3000 rpm for 5 minutes) to separate the target compounds from the n-hexane oil phase into the lower methanol-water phase, leveraging their hydrophilicity. This removes over 90% of lipid interfering compounds, such as triglycerides. The collected lower layer is concentrated to less than 3mL using nitrogen purge, achieving an enrichment factor of 3-5 times and a limit of detection (LOD) as low as 0.01μg / kg. The concentrate is then directly loaded onto an HLB solid-phase extraction column, eliminating the need for solvent exchange (e.g., evaporation and reconstitution), minimizing target compound loss and achieving a recovery rate of ≥85%.

[0047] Furthermore, in step S102, the polystyrene divinylbenzene pyrrolidone solid phase extraction column was pre-activated with 6 mL of methanol and 6 mL of pure water, washed with 6 mL of pure water and dried, and then the target substance was eluted with 10 mL of methanol-dichloromethane mixed solvent, and the eluate was collected. In the methanol-dichloromethane mixed solvent, the volume ratio of methanol to dichloromethane was 1:1.

[0048] Specifically, methanol activation of a polystyrene divinylbenzene pyrrolidone solid-phase extraction column fully wets the column packing, removes impurities adsorbed on the packing surface, and fully exposes the polar groups of the packing pyrrolidone, enhancing the adsorption capacity of the target compound. After activation, the column's static adsorption capacity for bisphenols and alkylphenol compounds increases by 20-30%, ensuring efficient retention of the target compound during sample loading. Methanol has a moderate polarity, which allows for sufficient wetting of the packing without excessively dissolving the target compound, preventing premature elution.

[0049] Furthermore, the HLB filler's hydrophilic-lipophilic balance preferentially adsorbs bisphenols and alkylphenols, while removing plasticizers such as phthalates and antioxidants such as BHT from polypropylene migration solutions, achieving a purification efficiency of ≥85%. HLB filler also achieves a removal rate of ≥95% for trace lipid interferences, such as triglycerides and fatty acids, from an olive oil simulant, improving the mass spectrometry signal-to-noise ratio by 3-5 times.

[0050] Specifically, washing the activated column with 6 mL of pure water can remove residual methanol and adjust the environment of the column packing to the aqueous phase, so that the target forms a stronger hydrogen bond or van der Waals force with the hydrogen bond receptor of the pyrrolidone of the packing when loading. After cleaning, the selectivity of the column for the target is significantly improved, and non-polar impurities such as lipids and pigments are preferentially eluted during the loading process, reducing matrix interference. The pure water washing step is simple, low-cost, and has no loss to the target. By draining the column, residual water can be removed, avoiding changes in the solubility of the target or weakening of the hydration of the surface groups of the packing due to the presence of water during the loading process. After drying, the retention efficiency of the column for the target is increased by 10-15%, which is suitable for the enrichment of low-concentration samples. The drying step can prevent the subsequent elution solvent from being diluted, ensuring elution efficiency. In addition, low-speed activation ensures uniform swelling of the packing, avoids cracking of the column bed or the formation of flow channels, and improves adsorption consistency.

[0051] Specifically, methanol, as a polar organic solvent, can interact with polar groups in the target molecules, such as hydrogen bonds; dichloromethane is a non-polar organic solvent and has good solubility for the non-polar parts of the target molecules, such as the benzene ring structure in bisphenol compounds and the alkyl chain in alkylphenol compounds. Since bisphenols and alkylphenol compounds have medium polarity, the polarity of the methanol-dichloromethane mixed solvent is highly matched with the target, which can not only dissolve the target, but also destroy its interaction with the surface groups of the filler, such as hydrogen bonds, to achieve efficient elution.

[0052] Compared to a single solvent such as pure methanol or pure dichloromethane, the mixed solvent elution efficiency is increased by 40-60%, and the recovery rate of the target compound reaches 92-98%. Optionally, in some embodiments, when eluting bisphenol A, the elution amount of the 1:1 mixed solvent (15 μg) is significantly higher than that of pure methanol (8 μg) and pure dichloromethane (6 μg).

[0053] Specifically, methanol solution can elute bisphenol compounds, and dichloromethane can elute alkylphenol compounds. The target compound can be obtained by eluting with methanol first and then with dichloromethane.

[0054] Specifically, during the desorption process using a polystyrene divinylbenzene pyrrolidone solid-phase extraction column, methanol achieved an elution efficiency of over 90% for bisphenols, while only 30-40% for alkylphenols. Dichloromethane could only remove trace amounts of bisphenols and 80% of alkylphenols. Testing revealed that a mixture of the two solvents achieved desorption efficiencies exceeding 90% for the analytes.

[0055] Optionally, in some embodiments, the addition of dichloromethane can reduce the polarity of the solvent and reduce the solubility of polar impurities such as sugars and amino acids, while the addition of methanol can maintain the solubility of the target, thereby eluting the target while avoiding a large amount of co-elution of impurities. The impurity content in the eluent is reduced by more than 50%, the matrix effect of subsequent liquid chromatography-tandem mass spectrometry analysis is significantly weakened, and the signal-to-noise ratio is improved by 2-3 times. In addition, the boiling points of methanol and dichloromethane are 64.7°C and 39.8°C, respectively, and the volatility of the mixed solvent is moderate. It can be easily concentrated to a suitable volume such as 1 mL by nitrogen blowing or rotary evaporation, and the target loss rate during the concentration process is <5%.

[0056] Specifically, the concentrated eluate can be directly sampled and analyzed without further processing, making it suitable for high-throughput analysis. Compared with high-boiling-point solvents (such as DMF and DMSO), mixed solvents offer higher concentration efficiency and less instrument contamination. The low impurity content in the eluate significantly reduces matrix effects during liquid chromatography-tandem mass spectrometry analysis, improving quantitative accuracy. In the analysis of olive oil extracts, the matrix effect after elution with mixed solvents was reduced from 30% compared to pure methanol to <10%, resulting in more reliable quantitative results.

[0057] Furthermore, the mass spectrometry conditions adopted the multiple reaction monitoring mode, and the ion source was the electrospray negative ion mode.

[0058] Specifically, Multiple Reaction Monitoring (MRM) mode uses two-stage mass spectrometry screening to monitor only the specific precursor ion to daughter ion transitions of the target compound, eliminating interfering signals such as isomers and matrix fragments. By focusing on specific ion pairs, MRM reduces signal dilution from non-target ions, achieving a limit of detection (LOD) of 0.005-0.1 g / kg, meeting the requirements of trace migration analysis. Compared to full scan or selected ion monitoring (SIM) modes, MRM offers 10-100 times higher sensitivity.

[0059] Optionally, in some embodiments, in the multiple reaction monitoring mode, a single run can simultaneously monitor the MRM channels of 8 environmental endocrine disruptors, such as bisphenol A / B / F, tetrachlorobisphenol A, octylphenol, nonylphenol, etc., with an efficiency improvement of 3-5 times.

[0060] Specifically, bisphenols and alkylphenols contain phenolic hydroxyl (-OH) or carbonyl (C=O) groups, which readily lose protons to form negative ions such as [MH]⁻ in negative electrospray ionization mode (ESI⁻). This ionization efficiency is significantly higher than in positive ionization mode (ESI⁺). This mode is suitable for the analysis of polar compounds containing phenolic hydroxyl or carbonyl groups, eliminating the need for derivatization and simplifying the process. In ESI⁻ mode, the target compound primarily forms [MH]⁻ ions, while matrix components (such as lipids and salts) tend to form [M+Cl]⁻ or [M+HCOO]⁻ adducts. MRM mode can be used to screen for characteristic ion transitions of the target compound and avoid matrix interference. Combined with a weakly acidic methanol / 5 mmol / L ammonium acetate mobile phase, ESI⁻ mode stably maintains the deprotonated state of the target compound, improving reproducibility (retention time RSD ≤ 0.1%).

[0061] Specifically, the MRM mode uses two-stage mass spectrometry to screen the characteristic ion transitions of the target. In combination, the ESI⁻ mode improves the response value by optimizing the ionization efficiency, controlling the matrix effect (ion suppression / enhancement) within ±15% (up to ±50% without correction), and the intra-day / inter-day precision (RSD) ≤5%. The combination of the two can achieve accurate detection of trace targets.

[0062] The beneficial effects of the present invention are as follows: 1) This method is based on a conventional liquid chromatography-tandem mass spectrometry platform, eliminating the need for ultra-high performance liquid chromatography (ULPL) systems and reducing laboratory equipment costs. By introducing an internal standard with similar properties to the target compound, this method can correct for systematic errors such as instrument fluctuations and injection volume differences, ensuring the accuracy of migration calculations. Solid-phase extraction cleanup eliminates matrix interference, making it particularly suitable for corporate R&D, third-party testing, and quality control by regulatory agencies.

[0063] 2) The methanol-dichloromethane mixed solvent used in the present invention has a high polarity match with the target compound, enabling efficient elution. The solid-phase extraction column used has a high recovery rate for substances such as bisphenols and alkylphenols, and has a long service life. This can improve the intensity and accuracy of the target compound signal in subsequent detection, facilitating more accurate content determination.

[0064] 3) The response of the isotopic internal standard of this invention is synchronized with that of the target compound. Internal standard correction can reduce errors in target compound signal variation caused by injection volume deviation, ion source stability, or chromatographic column performance fluctuations in liquid chromatography-tandem mass spectrometry analysis, thereby improving the repeatability and accuracy of quantitative results. The high sensitivity and stability of the isotopic internal standard can reduce the number of repeated analyses, and long-term use can reduce overall testing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 It is the multiple reaction monitoring (MRM) chromatogram of the bisphenol compound and the alkylphenol compound of the present invention. DETAILED DESCRIPTION

[0066] In order to make those skilled in the art more clearly understand the technical solutions of the present invention, the following examples are given for illustration. Unless otherwise specified, the raw materials, reagents or devices used in the following embodiments can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0067] A method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials using an internal standard method comprises the following steps: Step S101, "Sample Pretreatment": Cut the polypropylene material into 5 cm x 5 cm sample blocks, add an internal standard solution to a simulated immersion solution for migration testing, and use a 4% by volume acetic acid aqueous solution, a 10% by volume ethanol aqueous solution, or olive oil as the simulated immersion solution to obtain a solution to be purified; Step S102, solid phase extraction purification: the liquid to be purified is transferred to a solid phase extraction column, washed with pure water and drained, the target compound is eluted with a methanol-dichloromethane mixed solvent, and the eluate is collected; Step S103, sample volume adjustment and filtration: the eluate was concentrated to near dryness by nitrogen purge, the volume was adjusted to 1 mL with methanol-5 mmol / L ammonium acetate solution, and filtered through a 0.22 μm nylon microporous membrane; Step S104, instrumental analysis and quantification: liquid chromatography-tandem mass spectrometry is used for detection, and the migration amount of the target substance is calculated by the internal standard method based on pre-established standard curves for bisphenols and alkylphenols.

[0068] The environmental endocrine disrupting substances measured in polypropylene materials include bisphenol A, bisphenol B, bisphenol F, tetrachlorobisphenol A, octylphenol, 4-t-octylphenol, nonylphenol, and 4-nonylphenol.

[0069] The internal standard solution in step S101 is an isotope-labeled bisphenol and / or alkylphenol compound, including bisphenol A-d4, bisphenol Bd 10 , bischlorobisphenol A- 13 C 12 , octylphenol-d 17 ,4-t-octylphenol- 13 C6, nonylphenol-d5.

[0070] The extraction process of the olive oil test in step S101 includes adding 10 mL of n-hexane and vortex mixing, then adding 10 mL of methanol-water solution for back extraction, and transferring the target product to the lower layer of methanol-water solution.

[0071] The solid phase extraction column in step S102 is a polystyrene divinylbenzene pyrrolidone solid phase extraction column or a solid phase extraction column of model HLB-M-50. The activation condition of the solid phase extraction column is to sequentially pass 6 mL of methanol and pure water through the column at a flow rate of 1-2 mL / min.

[0072] The HLB-M-50 solid-phase extraction column was selected from Biocomma. Biocomma's HLB solid-phase extraction column features a high surface area, strong adsorption capacity, high analyte retention, minimal breakthrough, and a small elution volume. Its adsorption capacity and sample capacity are 3-10 times higher than those of C18 bonded silica gel. Biocomma's HLB solid-phase extraction column has a surface area of 600 m² / g, a uniform particle size of 40 μm, and an average pore size of 300 Å.

[0073] The preparation method of polystyrene divinylbenzene pyrrolidone as an adsorbent comprises the following steps: T1. Dissolve 1 g of methacrylic acid and 0.3 g of fatty alcohol polyoxyethylene-polyoxypropylene ether CPE-1500 in 100 ml of an aqueous solution containing 20% ethanol, add 0.3 g of polyvinyl pyrrolidone and 0.1 g of NaCl in sequence and stir evenly, add 10 g of styrene in a semi-continuous addition manner and stir to form an emulsion, dissolve 0.1 g of potassium persulfate in 10 ml of water at a dropping rate of 1 mL / min, heat to 95°C under nitrogen protection, stir for 2 hours, centrifuge, wash, and then dry to obtain polystyrene microspheres; T2. Disperse 7 g of polystyrene microspheres in 200 ml of 1% polyvinyl alcohol solution, sonicate for 30 minutes, mix 3 g of divinylbenzene and 30 ml of a porogen (toluene and n-heptane in a volume ratio of 1:2), slowly add the mixture dropwise to the microsphere dispersion, stir at room temperature for 12 hours to allow swelling, heat to 70°C, add 0.3 g of azobisisobutyronitrile to initiate a crosslinking reaction, continue for 8 hours, cool, filter, and extract with acetone Soxhlet for 24 hours to remove the porogen, and vacuum dry to obtain crosslinked polystyrene microspheres; T3. Disperse 10 g of cross-linked polystyrene microspheres in 200 ml of a 3:1 ethanol / water mixture. Add 2 g of N-vinyl pyrrolidone and 0.5 g of benzoyl peroxide. Purge with nitrogen for 30 minutes and react at 65°C for 12 hours. Filter, wash with methanol to remove unreacted NVP, and vacuum dry to obtain polystyrene divinylbenzene pyrrolidone.

[0074] The liquid chromatography conditions in step S104 are: C18 chromatographic column, mobile phase of methanol and 5 mmol / L ammonium acetate solution gradient elution.

[0075] The method for establishing a standard curve includes: preparing a standard solution containing gradient concentrations of bisphenols, alkylphenol compounds and a fixed concentration of an internal standard, performing linear fitting with the peak area ratio of the target substance to the internal standard as the ordinate and the concentration as the abscissa to obtain a standard curve, and substituting the measured peak area ratio of the target substance to the internal standard into the standard curve equation to achieve accurate quantification of the analyte.

[0076] In step S101, in the acetic acid and ethanol aqueous solution tests, 50 mL of the simulated liquid and the internal standard solution were added, and the mixture was thermostated at 100° C. for 30 minutes and then cooled. In the olive oil test, 50 g of olive oil and the internal standard solution were added, and after thermostating, 10 g of the oil sample was mixed with n-hexane and methanol-water solution. After centrifugation and stratification, the lower layer solution was collected, concentrated to less than 3 mL by nitrogen blowing, and cooled for purification.

[0077] In step S102, the polystyrene divinylbenzene pyrrolidone solid phase extraction column is pre-activated with 6 mL of methanol and 6 mL of pure water, washed with 6 mL of pure water and dried, and then the target substance is eluted with 10 mL of methanol-dichloromethane mixed solvent, and the eluate is collected. In the methanol-dichloromethane mixed solvent, the volume ratio of methanol to dichloromethane is 1:1.

[0078] Furthermore, the mass spectrometry conditions adopted the multiple reaction monitoring mode, and the ion source was the electrospray negative ion mode.

[0079] Based on the above content, the following are specific embodiments of the present invention: Example 1 Methanol, dichloromethane, and anhydrous ethanol were all chromatographically pure (Merck, USA); olive oil (AR grade, Guangzhou Reagent Factory); and deionized water filtered through a Milli-Q purification system. Bisphenol A, bisphenol B, bisphenol F, tetrachlorobisphenol A, octylphenol, 4-t-octylphenol, nonylphenol, and 4-nonylphenol were all from Dr. Ehrenstorfer, Germany; bisphenol A-d4, bisphenol Bd 10 , bischlorobisphenol A- 13 C 12 , octylphenol-d 17 ,4-t-octylphenol- 13 C6, nonylphenol-d5, both from Tianjin Alta Technology Co., Ltd.

[0080] Instruments and Equipment An Agilent 1290-6470 high-performance liquid chromatograph coupled with a triple quadrupole mass spectrometer was used; the chromatographic column was an Agilent Eclipse Plus C18 (2.1 m × 100 mm, 1.8 µm); a nitrogen blowdown system was used (ATR, USA); a high-speed centrifuge was used (Sigma, Germany); and an HLB solid-phase extraction cartridge (200 mg, 6 mL, Biocomma Biotech, model: HLB-M-50, adsorbent surface area: 600 m² / g, uniform particle size: 40 μm, average pore size: 300 Å).

[0081] Analysis conditions Chromatographic conditions: column temperature, 40°C; injection volume, 10 µL; mobile phase A, 5 mmol / L ammonium acetate solution; mobile phase B, methanol; mobile phase flow rate and gradient are shown in Table 1.

[0082] Table 1 Mobile phase time gradient

[0083] Mass spectrometry conditions: negative ion electrospray mode (-ESI); sheath gas temperature: 300°C; sheath gas flow rate: 12 L / min; drying gas temperature: 270°C; drying gas flow rate: 8 L / m; nebulizer gas pressure: 37 psi; capillary voltage: 3700 V; mass spectrometry conditions in multiple reaction monitoring mode are shown in Table 2.

[0084] Table 2 Mass spectrometry parameters in multiple reaction monitoring mode

[0085] Linear range and limit of quantitation Mixed standard solutions and corresponding internal standard mixed standard solutions were added to water, 4% acid solution, and 10% ethanol solutions at varying concentrations. Pretreatment was performed using optimized solid-phase extraction conditions. Liquid chromatography-mass spectrometry was used for determination. BPF was quantified using BPA-d4 as the internal standard, and 4-nonylphenol was quantified using nonylphenol-d5 as the internal standard. A standard curve was constructed using the quantification ion peak area as the ordinate and the concentration as the abscissa. The limit of quantification (LOQ) was determined using a 10x signal-to-noise ratio (S / N). The linear equation and LOQ are shown in Table 3.

[0086] Table 3 Standard curve parameters and quantification limits of bisphenols BPs and alkylphenols APs

[0087] Detection example: According to the established test method, 20 commercially available self-heating rice containers and 13 self-heating hot pot containers were tested. Of the 33 samples, 8 contained bisphenol A, 6 contained TCBPA, 4 contained 4-t-OP, 3 contained 4-OP, and 24 contained NPs. The results showed that the migration levels of each migrating substance in 4% aqueous solution were higher than those in 10% ethanol solution. The target substances were not detected in the olive oil immersion solution of the positive samples, demonstrating that neither BPs nor APs can be migrated out by oil. According to national limits, the amount of bisphenol A that can be released should not exceed 0.05 mg / kg. However, two samples tested exceeded this limit. The detection rate for NPs was as high as 72%, with levels ranging from 10 to 234 mg / kg.

[0088] like Figure 1 As shown, the present invention proposes a method for quantitatively analyzing the migration content of environmental endocrine disrupting substances (bisphenols and alkylphenols) in polypropylene materials using an internal standard method. The method comprises the following steps: setting migration parameters for a 4% acetic acid-water simulated solution, a 10% ethanol-water simulated solution, and an olive oil simulated solution; setting parameters for the solid-phase extraction cleanup of the migration solution; setting parameters for the concentration and reconstitution of the cleanup solution; and setting parameters for liquid chromatography-mass spectrometry analysis of the test solution. This method utilizes the specific retention mechanism of solid-phase extraction to separate interferences from the matrix. The highly selective ion channel monitoring function of the liquid chromatography-mass spectrometry instrument accurately identifies specific components in complex matrices based on the mass-to-charge ratio differences of the target compound's characteristic ion pairs. Combined with isotopic internal standard quantification, this method effectively ensures accurate quantification of trace components of bisphenols and alkylphenols that migrate in polypropylene materials. This method offers simple pretreatment for the determination of the migration of bisphenols and alkylphenols in polypropylene materials, high qualitative analysis accuracy, and excellent reproducibility for trace determination, making it highly applicable.

[0089] Example 2 1. Target compound selection Bisphenols: bisphenol A, bisphenol B, bisphenol F, tetrachlorobisphenol A.

[0090] Alkylphenols: octylphenol, 4-t-octylphenol, nonylphenol, 4-nonylphenol.

[0091] Internal standards: bisphenol A-d4, bisphenol B-d10, bischlorobisphenol A-13C12, octylphenol-d17, 4-t-octylphenol-13C6, nonylphenol-d5.

[0092] 2. Solution preparation Stock solution (1000 μg / mL): Weigh 10 mg of each target compound and internal standard, dissolve them in methanol and dilute to 10 mL.

[0093] Mixed intermediate solution (10 μg / mL): Take 100 μL of each stock solution and dilute to 10 mL with methanol.

[0094] Internal standard working solution (1 μg / mL): Take 100 μL of the internal standard stock solution and dilute it to 100 mL with methanol.

[0095] Matrix Standard Mix: The mixed intermediate solution was added to a blank matrix (4% acetic acid aqueous solution, 10% ethanol aqueous solution or olive oil) to prepare five concentration levels (0.5, 1, 5, 10, 50 ng / mL).

[0096] A fixed amount of internal standard (10 ng / mL) was added to each concentration to ensure that the peak area ratio of the internal standard to the target compound was within a reasonable range.

[0097] The sample pretreatment was consistent with the above operation.

[0098] Recovery rate (%) = (C 实测 xV 定容 ) / (C 添加 xV 取样 ) x 100% Among them, C 实测 is the concentration calculated by the standard curve, V 定容 is the final reconstitution volume (1 mL), C 添加 is the spike concentration (10 ng / mL), V 取样 is the sampling volume (10 mL).

[0099] Each adsorbent was tested 5 times, and the average recovery rate was calculated.

[0100] In this embodiment, the solid phase extraction column used is a polystyrene divinylbenzene pyrrolidone solid phase extraction column, the adsorbent specific surface area is 850m² / g, the uniform particle size is 60μm, and the average pore size is 300Å.

[0101] Example 3 The difference between Example 3 and Example 2 is that the solid phase extraction column used is Biocomma, model: HLB-M-50, 200 mg, 6 mL, adsorbent specific surface area: 600 m² / g, uniform particle size: 40 μm, average pore size: 300 Å.

[0102] Example 4 The difference between Example 4 and Example 2 is that the solid phase extraction column used is an Oasis HLB solid phase extraction column (Waters, model: WAT094226, 60 mg, 3 mL).

[0103] Example 5 The difference between Example 5 and Example 2 is that the solid phase extraction column used is a C18 solid phase extraction column (Thermo Fisher, model: 60108-305-P, 500 mg, 6 mL).

[0104] Example 6 The difference between Example 6 and Example 2 is that the solid phase extraction column used is a PSA solid phase extraction column (manufactured by Jinlan, 100 mg, 3 mL).

[0105] Example 7 The difference between Example 7 and Example 2 is that the eluent is a methanol solution.

[0106] Comparative Example 8 The difference between Example 8 and Example 2 is that dichloromethane solution is used as the eluent.

[0107] Table 4 Recovery statistics of Example 2 to Example 8

[0108] With respect to Examples 2 to 8, the polystyrene divinylbenzene pyrrolidone solid phase extraction column of Example 2 generally exhibits a higher recovery rate due to its good balance between hydrophobicity and hydrophilicity.

[0109] The HLB-M-50 solid-phase extraction column from Biocomma in Example 3 also exhibited a high recovery rate due to its balanced hydrophilicity and hydrophobicity. The Oasis HLB solid-phase extraction column in Example 4 had a high adsorption capacity for both polar and non-polar compounds and exhibited a high recovery rate, but was slightly lower than the polystyrene divinylbenzene pyrrolidone solid-phase extraction column.

[0110] The C18 solid phase extraction column of Example 5 is mainly suitable for compounds with strong hydrophobicity, and the recovery rate of bisphenols is slightly low.

[0111] The PSA solid phase extraction column of Example 6 is mainly used to remove acidic impurities, and the recovery rate of bisphenols and alkylphenols is relatively low.

[0112] In Examples 7 and 8, during the desorption process using a polystyrene divinylbenzene pyrrolidone solid-phase extraction column, methanol achieved an elution efficiency of over 90% for bisphenols, while only 30-40% for alkylphenols. Dichloromethane could only elute trace amounts of bisphenols and 80% of the alkylphenols. Testing showed that a mixture of the two solvents achieved an elution desorption efficiency of over 90% for the analytes.

[0113] This method, based on a conventional liquid chromatography-tandem mass spectrometry platform, eliminates the need for an ultra-high performance liquid chromatography system, reducing laboratory equipment investment costs. By introducing an internal standard with similar properties to the target compound, the method can correct for systematic errors such as instrument fluctuations and injection volume differences, ensuring the accuracy of migration calculations. Solid-phase extraction cleanup eliminates matrix interference, making it particularly suitable for corporate R&D, third-party testing, and quality control by regulatory agencies.

[0114] The methanol-dichloromethane mixed solvent used in the present invention has a high polarity match with the target object and can achieve efficient elution. The solid-phase extraction column used has a high recovery rate for substances such as bisphenols and alkylphenols, and has a long service life. It can improve the intensity and accuracy of the target object signal in subsequent detection, which helps to more accurately determine the content.

[0115] The isotopic internal standard of the present invention synchronizes its response with that of the target. Internal standard correction can reduce errors in target signal variation caused by injection volume deviation, ion source stability, or chromatographic column performance fluctuations in liquid chromatography-tandem mass spectrometry analysis, thereby improving the repeatability and accuracy of quantitative results. The high sensitivity and stability of the isotopic internal standard can reduce the number of repeated analyses, and long-term use can reduce overall testing costs.

[0116] The above examples are merely provided to further illustrate the technical content of the present invention for easier understanding by the reader, but do not limit the embodiments of the present invention to these examples. Any extension or re-creation of the technology based on the present invention is protected by the present invention. The scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials using an internal standard method, characterized in that: The steps include: Step S101, "Sample Pretreatment": Cut the polypropylene material into sample blocks, add an internal standard solution to a simulated immersion solution to perform a migration test, and use a 4% by volume aqueous acetic acid solution, a 10% by volume aqueous ethanol solution, or olive oil as the simulated immersion solution to perform the migration test to obtain a solution to be purified; Step S102, solid phase extraction purification: the liquid to be purified is transferred to a solid phase extraction column, washed with pure water and drained, the target compound is eluted with a methanol-dichloromethane mixed solvent, and the eluate is collected; Step S103, sample volume adjustment and filtration: the eluate is concentrated to near dryness by nitrogen purge, the volume is adjusted to 1 mL with methanol-5 mmol / L ammonium acetate solution, and filtered through a nylon microporous filter membrane; Step S104, instrumental analysis and quantification: liquid chromatography-tandem mass spectrometry is used for detection, and the migration amount of the target substance is calculated by the internal standard method based on pre-established standard curves for bisphenols and alkylphenols.

2. A method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials using an internal standard method according to claim 1, characterized in that: The environmental endocrine disrupting substances measured in polypropylene materials include bisphenol A, bisphenol B, bisphenol F, tetrachlorobisphenol A, octylphenol, 4-t-octylphenol, nonylphenol, and 4-nonylphenol.

3. A method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials by an internal standard method according to claim 1, characterized in that: The internal standard solution in step S101 is an isotope-labeled bisphenol and / or alkylphenol compound, including bisphenol A-d4, bisphenol Bd 10 , bischlorobisphenol A- 13 C 12 , octylphenol-d 17 ,4-t-octylphenol- 13 C6, nonylphenol-d5.

4. A method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials by an internal standard method according to claim 1, characterized in that: The extraction process of the olive oil test in step S101 includes adding 10 mL of n-hexane and vortex mixing, then adding 10 mL of methanol-water solution for back extraction, and transferring the target product to the lower layer of methanol-water solution.

5. A method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials by an internal standard method according to claim 1, characterized in that: The solid phase extraction column in step S102 is a polystyrene divinylbenzene pyrrolidone solid phase extraction column or a solid phase extraction column of model HLB-M-50. The activation condition of the solid phase extraction column is to sequentially pass 6 mL of methanol and pure water through the column at a flow rate of 1-2 mL / min.

6. A method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials by an internal standard method according to claim 1, characterized in that: The liquid chromatography conditions in step S104 are: C18 chromatographic column, mobile phase of methanol and 5 mmol / L ammonium acetate solution gradient elution.

7. A method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials by an internal standard method according to claim 1, characterized in that: The method for establishing a standard curve includes: preparing a standard solution containing gradient concentrations of bisphenols, alkylphenol compounds and a fixed concentration of an internal standard, performing linear fitting with the peak area ratio of the target substance to the internal standard as the ordinate and the concentration as the abscissa to obtain a standard curve, and substituting the measured peak area ratio of the target substance to the internal standard into the standard curve equation to achieve accurate quantification of the analyte.

8. The method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials by internal standard method according to claim 1, characterized in that: In step S101, in the acetic acid and ethanol aqueous solution tests, 50 mL of the simulated liquid and the internal standard solution were added, and the mixture was thermostated at 100° C. for 30 minutes and then cooled. In the olive oil test, 50 g of olive oil and the internal standard solution were added, and after thermostating, 10 g of the oil sample was mixed with n-hexane and methanol-water solution. After centrifugation and stratification, the lower layer solution was collected, concentrated to less than 3 mL by nitrogen blowing, and cooled for purification.

9. The preparation method of a method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials by internal standard method according to claim 1, characterized in that: In step S102, the polystyrene divinylbenzene pyrrolidone solid phase extraction column is pre-activated with 6 mL of methanol and 6 mL of pure water, washed with 6 mL of pure water and dried, and then the target substance is eluted with 10 mL of methanol-dichloromethane mixed solvent, and the eluate is collected. In the methanol-dichloromethane mixed solvent, the volume ratio of methanol to dichloromethane is 1:

1.

10. The preparation method of a method for quantitatively analyzing the migration content of bisphenols and alkylphenols in polypropylene materials by internal standard method according to claim 1, characterized in that: The mass spectrometry conditions adopted the multiple reaction monitoring mode and the ion source was the electrospray negative ion mode.

Images