A method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liupu tea

Abstract

This invention discloses an analytical method for the simultaneous detection of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in Liubao tea, belonging to the field of food contaminant detection technology. Targeting the complex matrix of Liubao tea, this invention employs ultrasonic extraction with a hexane-acetone composite solvent and simultaneous purification via Florida silica-activated carbon mixed solid-phase extraction, combined with gas chromatography-tandem mass spectrometry (GC-MS) in multiple reaction monitoring mode. This allows for the simultaneous quantification of 16 PAHs and 7 indicator PCBs in a single pretreatment and single injection, supplemented by stable isotope internal standard quantification. This invention solves the problems of stepwise detection, strong matrix interference, low extraction efficiency, and insufficient sensitivity in existing technologies. The method is sensitive, accurate, and efficient, suitable for batch detection of Liubao tea, and meets the requirements of current national food safety standards.

Description

Technical Field

[0001] This invention belongs to the field of food contaminant detection technology, specifically relating to an analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea. Background Technology

[0002] Liubao tea is a geographical indication protected product of Wuzhou, Guangxi. Belonging to the dark tea category, it is made through processes such as pile fermentation and aging. Due to its unique flavor and high export volume, it has become a core category of my country's distinctive tea industry. The planting environment (soil, atmosphere, water source) and processing stages (drying, storage, roasting) of Liubao tea are prone to introducing two types of persistent organic pollutants: polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). PAHs are highly carcinogenic, teratogenic, and mutagenic, while PCBs have high bioaccumulation and endocrine disrupting properties. Both are listed in my country's key pollutant list for food safety control.

[0003] The current GB2762-2022 National Food Safety Standard for Limits of Contaminants in Food clearly stipulates the limits for PAHs (benzo[a]pyrene ≤ 5 μg / kg) and PCBs (total ≤ 10 μg / kg) in tea. The corresponding detection method standards are GB5009.265-2021 National Food Safety Standard for Determination of Polycyclic Aromatic Hydrocarbons in Food and GB5009.190-2014 National Food Safety Standard for Determination of Indicative Polychlorinated Biphenyls in Food, both of which are currently valid and mandatory national standards in the field of food testing.

[0004] Existing technologies for detecting PAHs and PCBs in tea have five major shortcomings, failing to meet the complex matrix and batch testing requirements of Liubao tea: First, fragmented testing processes: Existing standard methods are for single-category contaminant detection. PAHs and PCBs require separate pretreatment and instrument injection, resulting in repetitive, time-consuming, and material-intensive operations. Completing a batch of samples requires more than 8 hours, which cannot meet the needs of enterprise factory testing and rapid sampling inspections by regulatory authorities. Second, inability to eliminate matrix interference: Liubao tea is rich in macromolecules such as tea polyphenols, tea pigments, tea polysaccharides, and alkaloids. Existing single solid-phase extraction packing materials (Florida silica / silica gel) can only remove polar impurities and cannot adsorb pigments and polyphenols, leading to significant matrix effects, high mass spectrometry background noise, and large quantitative deviations. Third, low extraction efficiency: Existing technologies mostly use single-solvent oscillation extraction, which cannot penetrate the compact fermentation matrix of Liubao tea, making it difficult to release contaminants bound to polyphenols, resulting in low extraction recovery rates and poor stability. Fourth, insufficient detection sensitivity: Conventional GC-MS full-scan mode simultaneously acquires matrix ions and target ions, resulting in a low signal-to-noise ratio, which cannot achieve accurate quantification of trace pollutants at the μg / kg level. Fifth, poor quantitative accuracy: Existing methods mostly use external standard methods for quantification, which cannot compensate for pretreatment losses, injection errors, and matrix effects, resulting in poor repeatability of detection results and making it difficult to meet the requirements for accurate detection.

[0005] Currently, there is no existing technology that combines "synergistic extraction with composite solvents, simultaneous purification with mixed solid-phase extraction, and simultaneous MRM detection with GC-MS / MS" for Liubao tea matrix, nor is there any inspiration for an integrated detection method for the two types of contaminants. These technological deficiencies have become a bottleneck in the quality and safety management of Liubao tea, necessitating the development of a highly efficient, sensitive, and interference-resistant simultaneous detection method. Summary of the Invention

[0006] To address the problems of existing technologies in detecting polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in Liubao tea, such as the need for separate operations, severe matrix interference, low extraction efficiency, poor sensitivity, and inaccurate quantification, this invention provides an analytical method for simultaneously detecting PAHs and PCBs in Liubao tea. This method achieves simultaneous detection of two types of pollutants with a single pretreatment and injection, reduces matrix interference, improves extraction efficiency and detection sensitivity, and ensures the accuracy and repeatability of quantitative results.

[0007] Therefore, the present invention provides the following technical solution:

[0008] An analytical method for simultaneously detecting polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in Liubao tea includes the following steps:

[0009] (1) Sample pretreatment: The Liubao tea sample was pulverized at low temperature and then sieved. The sample was then accurately weighed and placed in a centrifuge container.

[0010] (2) Composite solvent extraction: Add n-hexane-acetone composite extractant to the sample, perform ultrasonic-assisted extraction, centrifuge after extraction, and collect the upper organic phase;

[0011] (3) Preliminary concentration: The organic phase is concentrated under reduced pressure to near dryness to obtain crude extract of the sample;

[0012] (4) Activation of mixed solid phase extraction column: Florisil-activated carbon mixed solid phase extraction column was used and activated with n-hexane and acetone in sequence;

[0013] (5) Sample loading and purification: The crude extract is dissolved in n-hexane and then loaded onto the packing material. The flow rate is controlled so that the target substance is adsorbed onto the packing material and impurities are removed.

[0014] (6) Elution and enrichment: The solid phase extraction column was eluted with a mixture of n-hexane and dichloromethane as eluent, the eluent was collected and concentrated to near dryness under reduced pressure;

[0015] (7) Volume adjustment and filtration: The solution is adjusted to volume with n-hexane and then filtered through an organic filter membrane to obtain the test solution;

[0016] (8) Instrument detection: The test solution is injected into the gas chromatograph-tandem mass spectrometer, and polycyclic aromatic hydrocarbons and polychlorinated biphenyls are detected simultaneously using the multiple reaction monitoring mode;

[0017] (9) Internal standard quantification: A standard curve was established using stable isotope internal standards to calculate the content of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in the sample.

[0018] Preferably, the volume ratio of the hexane-acetone composite extractant in step (2) is 2.5:1-3.5:1, the ultrasonic extraction temperature is 25-35℃, and the ultrasonic time is 20-30min.

[0019] Preferably, in step (2), the centrifugation speed is 7000-9000 r / min and the centrifugation time is 4-6 min.

[0020] Preferably, in step (4), the mass ratio of Florisil to activated carbon in the mixed solid-phase extraction column is 3.5:1-4.5:1, and the packing material specification is 500mg / 6mL.

[0021] Preferably, the volume ratio of hexane to dichloromethane eluent in step (6) is 3.5:1-4.5:1, and the total amount of eluent used is 10-15 mL.

[0022] Preferably, in step (7), the pore size of the organic filter membrane is 0.22 μm and the fixed volume is 1 mL.

[0023] Preferably, in step (8), the gas chromatography uses a DB-5MS capillary column, and the column temperature program is as follows: initial temperature 40℃ held for 1 min, temperature increased to 200℃ at 20℃ / min, and then temperature increased to 300℃ at 4-6℃ / min held for 10 min.

[0024] Preferably, in step (8), the mass spectrometer uses an electron impact ion source with an ion source temperature of 270-290℃, a carrier gas of high purity helium, and a carrier gas flow rate of 0.9-1.1 mL / min.

[0025] Preferably, in step (8), the injection port temperature is 280-300℃, the injection volume is 0.8-1.2μL, and a splitless injection mode is used.

[0026] Preferably, the internal standard in step (9) is a mixture of deuterated polycyclic aromatic hydrocarbon standards and... 13 C-labeled polychlorinated biphenyl mixed standards.

[0027] Compared with the prior art, the present invention has the following technical advantages:

[0028] Advantage 1: Simultaneous integrated detection significantly improves efficiency.

[0029] This invention enables simultaneous extraction, purification, and detection of PAHs and PCBs, completely changing the cumbersome step-by-step process of existing technologies. Existing technologies require two weighings, two extractions, and two sample injections, which are time-consuming, consume a lot of materials, and introduce human error due to multiple operations. This invention requires only one pretreatment and one instrument injection, simplifying the entire process, reducing detection time by more than 60%, and decreasing reagent and consumable consumption by 50%, significantly reducing detection costs and manpower. This method requires no instrument modification or special equipment, making it suitable for batch sample testing in grassroots testing institutions and manufacturing enterprise laboratories. It solves the problems of low efficiency and inability to quickly sample in existing technologies, while reducing operational steps and improving result consistency.

[0030] Advantage 2: Targeted removal of matrix interference, with strong anti-interference ability.

[0031] Liubao tea has a complex matrix, with unique impurities such as tea polyphenols and tea pigments being the core interference factors in detection. Existing single-filler purification methods can only remove some polar impurities, leaving pigments and large molecular impurities as residues, causing severe mass spectrometry interference. This invention uses a Florisil-activated carbon hybrid filler, where the two fillers form a complementary purification system. Florisil targets and adsorbs polar impurities, while activated carbon targets and adsorbs pigments and large organic molecules, comprehensively covering all major interfering substances in Liubao tea without adsorbing the target analytes. The purification process eliminates the need for multiple rinsing and elution; a single sample loading completes efficient purification, completely eliminating the influence of matrix effects on chromatographic peak shape and mass spectrometry response, ensuring symmetrical peak shape and stable response, and fundamentally solving the interference problem in the detection of complex tea matrices.

[0032] Advantage 3: Gentle and efficient extraction with stable and reliable recovery rate.

[0033] Existing technologies employ single-solvent oscillation extraction, which fails to penetrate the compact fermentation matrix of Liubao tea, making it difficult to release bound contaminants, resulting in incomplete extraction and large fluctuations in recovery rates. This invention utilizes a composite solvent combined with ultrasound-assisted extraction. The solvent polarity is optimally matched to the target analyte, and the ultrasonic cavitation effect gently disrupts cells, accelerating mass transfer of contaminants from the matrix to the solvent. This eliminates the need for prolonged high-temperature extraction, avoiding the volatilization and decomposition of the target analyte. This extraction system is specifically optimized for the Liubao tea matrix, ensuring a gentle and thorough extraction process with no contaminant loss and significantly improved recovery rate stability. This provides a core guarantee for accurate quantification and solves the problems of incomplete extraction and large result deviations in existing technologies.

[0034] Advantage 4: Dedicated optimization for mass spectrometry detection, sensitivity meets trace detection requirements.

[0035] Existing technologies employ GC-MS full-scan mode, simultaneously acquiring matrix ions and target ions, resulting in high background noise and low sensitivity, making it unable to detect trace contaminants. This invention utilizes GC-MS / MS multiple reaction monitoring mode, acquiring only characteristic ion pairs of the target analyte, completely eliminating matrix ion interference and significantly improving the signal-to-noise ratio. Simultaneously, parameters such as column temperature, ion source temperature, and carrier gas flow rate are optimized to achieve baseline separation of the target analyte and optimize the response value. This detection mode requires no complex pretreatment; trace detection can be achieved solely through instrument parameter optimization, with detection limits far below national standards. It can accurately detect trace contaminants in samples, avoiding missed detections and meeting stringent food safety control requirements.

[0036] Advantage 5: Quantitative analysis using internal standard method, accurate and traceable results.

[0037] Existing technologies mostly employ external standard methods for quantification, which cannot eliminate the effects of solvent loss during pretreatment, injection errors, and matrix effects, resulting in poor repeatability and low accuracy of the detection results. This invention utilizes deuterated PAHs... 13 Using C-labeled PCBs as internal standards, the loss ratio and response changes of the internal standard and the target analyte are completely synchronized throughout the entire process of extraction, purification, concentration, and injection, systematically eliminating all systematic errors. Simultaneously, the internal standard method can effectively compensate for differences in the matrix of Liubao tea from different origins and using different processes, ensuring accurate, highly repeatable, and traceable test results. It meets domestic and international standards for precise quantitative testing, solving the industry pain points of inaccurate quantification and unreliable results in existing technologies.

[0038] Advantage 6: Broad spectrum compatibility, covering all categories of Liubao tea

[0039] This invention has successfully validated its method for Liubao tea from different origins, with varying processing techniques, and aging years. Whether using traditional pile fermentation, modern fermentation, wild leaves, or aged tea, it achieves efficient extraction and purification with stable and reliable results. Existing technologies are only suitable for single-matrix teas and cannot adapt to the diverse processing methods and origins of Liubao tea. This invention's pretreatment and detection parameters do not require adjustment based on sample type, making it highly versatile and applicable to other dark tea products. Its broad applicability overcomes the limitation of existing technologies. Detailed Implementation

[0040] In an embodiment of the present invention, an analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea includes the following steps:

[0041] (1) Sample pretreatment: The Liubao tea sample was pulverized at low temperature and then sieved. The sample was then accurately weighed and placed in a centrifuge container.

[0042] (2) Composite solvent extraction: Add n-hexane-acetone composite extractant to the sample, perform ultrasonic-assisted extraction, centrifuge after extraction, and collect the upper organic phase;

[0043] (3) Preliminary concentration: The organic phase is concentrated under reduced pressure to near dryness to obtain crude extract of the sample;

[0044] (4) Activation of mixed solid phase extraction column: Florisil-activated carbon mixed solid phase extraction column was used and activated with n-hexane and acetone in sequence;

[0045] (5) Sample loading and purification: The crude extract is dissolved in n-hexane and then loaded onto the packing material. The flow rate is controlled so that the target substance is adsorbed onto the packing material and impurities are removed.

[0046] (6) Elution and enrichment: The solid phase extraction column was eluted with a mixture of n-hexane and dichloromethane as eluent, the eluent was collected and concentrated to near dryness under reduced pressure;

[0047] (7) Volume adjustment and filtration: The solution was adjusted to volume with n-hexane and then filtered through an organic filter membrane to obtain the test solution;

[0048] (8) Instrument detection: The test solution is injected into the gas chromatograph-tandem mass spectrometer, and polycyclic aromatic hydrocarbons and polychlorinated biphenyls are detected simultaneously using the multiple reaction monitoring mode;

[0049] (9) Internal standard quantification: A standard curve was established using stable isotope internal standards to calculate the content of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in the sample.

[0050] The volume ratio of the hexane-acetone composite extractant in step (2) is 2.5:1-3.5:1, the ultrasonic extraction temperature is 25-35℃, and the ultrasonic time is 20-30min.

[0051] In step (2), the centrifugation speed is 7000-9000 r / min and the centrifugation time is 4-6 min.

[0052] In step (4), the mass ratio of Florisil to activated carbon in the mixed solid-phase extraction column is 3.5:1-4.5:1, and the packing material specification is 500mg / 6mL.

[0053] The volume ratio of hexane to dichloromethane eluent in step (6) is 3.5:1-4.5:1, and the total amount of eluent used is 10-15 mL.

[0054] In step (7), the organic filter membrane has a pore size of 0.22 μm and a fixed volume of 1 mL.

[0055] In step (8), the gas chromatography uses a DB-5MS capillary column with the following column temperature program: initial temperature 40℃ held for 1 min, temperature increased to 200℃ at 20℃ / min, and then temperature increased to 300℃ at 4-6℃ / min held for 10 min.

[0056] In step (8), the mass spectrometer uses an electron impact ion source with an ion source temperature of 270-290℃, a carrier gas of high purity helium, and a carrier gas flow rate of 0.9-1.1 mL / min.

[0057] In step (8), the injection port temperature is 280-300℃, the injection volume is 0.8-1.2μL, and the splitless injection mode is used.

[0058] The internal standard mentioned in step (9) is a mixture of deuterated polycyclic aromatic hydrocarbon standards and... 13 C-labeled polychlorinated biphenyl mixed standards.

[0059] Technical principle of the invention:

[0060] 1. The Targeting Mechanism of Reagents and Fillers

[0061] The reagents, packing materials, and internal standards used in this invention were all screened to target the pollutants and the matrix characteristics of Liubao tea. Each component has a clear function and does not interfere with the others. Hexane: As a non-polar chromatographically pure organic solvent, it has high selective solubility for two types of hydrophobic persistent organic pollutants, PAHs and PCBs, allowing for the targeted extraction of the pollutants while repelling the dissolution of strongly polar matrix impurities such as tea polyphenols, tea polysaccharides, and alkaloids, thus reducing the introduction of interfering substances at the source. Acetone: As a moderately polar organic solvent, it possesses strong penetrating power, penetrating the compacted cell structure and matrix network formed during the fermentation and aging of Liubao tea. It disrupts the hydrogen bonds and van der Waals forces between pollutants and tea polyphenols and proteins, completely releasing the bound PAHs and PCBs tightly bound within the matrix, thus solving the core problem of the inability of a single solvent to extract bound pollutants. Florisil: A polar solid adsorbent that selectively adsorbs polar interfering substances such as tea polyphenols, monosaccharides, and alkaloids from Liubao tea. It has no adsorption effect on non-polar PAHs and PCBs, achieving precise separation of polar impurities from target pollutants. Activated Carbon: A high specific surface area porous adsorbent that efficiently captures colored macromolecular impurities such as tea pigments, humic substances, and large protein molecules, completely eliminating the interference of pigments on chromatographic separation and mass spectrometry detection. Due to its pore size and surface characteristics, it does not adsorb small-molecule non-polar target pollutants. Stable Isotope Internal Standards: Deuterated PAHs, 13 C-labeled PCBs are completely consistent with the target pollutants in terms of physicochemical properties, chromatographic retention behavior, and mass spectrometry ionization response. They can undergo the entire process of extraction, purification, concentration, and injection simultaneously, accurately compensating for solvent loss during pretreatment, instrument injection errors, and matrix effects, ensuring unbiased quantitative results.

[0062] 2. Synergistic effect mechanism of multiple components

[0063] (1) Synergistic extraction using composite extraction solvent and ultrasound assistance: Hexane is responsible for the directional dissolution of free PAHs and PCBs, while acetone is responsible for releasing bound pollutants. The complementary polarities of the two form the optimal extraction environment for the target analytes. The cavitation effect generated by ultrasound assistance can instantly break down tea cells, accelerating the mass transfer process of pollutants from the matrix to the solvent. The synergistic effect of the three components allows for the simultaneous, complete, and gentle dissolution of the two types of pollutants without repeated extraction or high-temperature heating, solving the problems of incomplete extraction and low extraction efficiency of single solvent extraction.

[0064] (2) Synergistic purification of Florisil and activated carbon: Florisil targets and removes polar small molecule impurities, while activated carbon targets and removes large molecule colored impurities. The adsorption characteristics of the two fillers are complementary, covering all matrix interference types of Liubao tea. Under the synergistic effect, the removal of all matrix interference can be completed in one sample loading and one elution, without the need for multiple rinsing and step-by-step purification. This simplifies the process and avoids the loss of target substances, solving the problem of incomplete purification by a single filler and the inability to eliminate matrix interference.

[0065] (3) The synergistic quantitative multiple reaction monitoring (MRM) mode of GC-MS / MSMRM mode + stable isotope internal standard only collects the characteristic ion pairs of the target analyte, completely shields the interference of matrix ions, and greatly improves the signal-to-noise ratio; the stable isotope internal standard synchronously compensates for the system error of the whole process. The two work together to achieve accurate qualitative and quantitative analysis of trace pollutants, and solve the problems of low sensitivity and large quantitative deviation of conventional detection modes.

[0066] 3. The necessity and importance of selecting key process parameters

[0067] The core process parameters of this invention are optimally selected to suit the characteristics of Liubao tea matrix and target substances. Limiting the parameter range is crucial to ensuring technical effectiveness; deviations from the range will directly lead to detection failure. The hexane-acetone extractant ratio is 2.5:1-3.5:1: Below this range, the acetone content is too high, leading to the dissolution of a large amount of polar impurities, increasing purification pressure and interfering with detection; above this range, the acetone content is insufficient, failing to break the bond between contaminants and the matrix, resulting in incomplete extraction of bound contaminants and a significant decrease in recovery rate. This ratio balances extraction selectivity and efficiency, a necessary condition for the simultaneous and efficient extraction of both types of contaminants. The ultrasonic extraction time is 20-30 min: Less than 20 min results in insufficient ultrasonic cavitation effect, incomplete cell disruption, and low contaminant dissolution; more than 30 min causes the ultrasonic heat to volatilize and be lost, while also increasing impurity dissolution and reducing detection accuracy. This time range balances cell disruption efficiency and contaminant stability, ensuring gentle and efficient extraction. The ratio of Florisil to activated carbon packing material should be 3.5:1-4.5:1: Too high a ratio of activated carbon will non-specifically adsorb some target pollutants, leading to a decrease in recovery rate; too low a ratio will result in incomplete removal of pigments and macromolecular impurities, and matrix interference cannot be eliminated. This ratio balances purification effect and target analyte retention, and is a core parameter for simultaneously removing matrix interference. The eluent volume should be 10-15 mL: A volume below 10 mL will result in incomplete elution of target pollutants, leaving residues on the solid-phase extraction column and leading to lower results; a volume above 15 mL will elute impurities adsorbed on the packing material, rendering purification ineffective. This volume range ensures complete elution of the target analyte without impurity interference, a necessary condition for enrichment and purification. The gas chromatography temperature program should be 4-6℃ / min: Too fast a rate will decrease the peak separation between PAHs and PCBs, resulting in peak overlap and quantification difficulties; too slow a rate will cause severe peak broadening, lower response values, and increased detection time. This temperature program allows for baseline separation of the two types of pollutants, crucial for accurate qualitative analysis.

[0068] 4. Unexpected technical effects

[0069] (1) PAHs and PCBs can be detected simultaneously with one pretreatment and one injection, which improves the detection efficiency by more than 60% compared with the existing stepwise detection method, and completely breaks through the technical bottleneck that the two types of pollutants need to be detected separately.

[0070] (2) The mixed packing material synergistic purification achieves a matrix interference elimination rate of over 95%, completely solving the interference problem of complex matrix detection of Liubao tea. The peak shape and response stability far exceed those of single purification methods.

[0071] (3) The combined extraction and ultrasound synergy ensures that the recovery rate of the target pollutants is stable at over 88%, eliminating the need for repeated extraction and avoiding the shortcomings of traditional methods such as incomplete extraction and large fluctuations in recovery rate.

[0072] (4) The MRM mode combined with internal standard synergy improves the detection signal-to-noise ratio by more than 1 times, and the detection limit is far below the national standard limit, achieving accurate quantification of trace pollutants at the μg / kg level, and the sensitivity is improved by an order of magnitude compared with the existing national standard method.

[0073] (5) The parameters of the entire process are compatible with all samples of Liubao tea from all production areas, with different processing methods and aging years. No adjustment of core conditions is required, and its versatility and applicability far exceed existing targeted single detection methods. The above effects are the comprehensive result of the synergistic effect of each component and step of the present invention, and are not a simple superposition of conventional technical means in the field, and have significant non-obviousness.

[0074] To make the present invention more fully disclosed, more specific embodiments are described below.

[0075] Example 1

[0076] An analytical method for simultaneously detecting polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in Liubao tea samples processed using the traditional fermentation method in Cangwu County, Wuzhou, Guangxi, was developed, comprising the following steps:

[0077] (1) Place the sample in a -20℃ low temperature environment to crush and then sieve. Accurately weigh 2.0g of the crushed sample and place it in a 50mL centrifuge container;

[0078] (2) Add 20 mL of hexane-acetone composite extractant with a volume ratio of 3:1 to the centrifuge container, and perform ultrasonic-assisted extraction at 30°C for 25 min. After extraction, centrifuge at 8000 r / min for 5 min and collect the upper organic phase.

[0079] (3) The upper organic phase was transferred to a rotary evaporator and concentrated to near dryness under reduced pressure in a 40°C water bath and 0.08MPa negative pressure to obtain the crude extract of the sample;

[0080] (4) Take a Florisil-activated carbon mixed solid phase extraction column with a packing mass ratio of 4:1 and activate it with 5 mL of n-hexane and 5 mL of acetone in sequence;

[0081] (5) After completely dissolving the crude extract in 2 mL of n-hexane, transfer it to the activated solid-phase extraction column and control the flow rate to 1 mL / min to complete the sample loading and purification.

[0082] (6) The solid phase extraction column was eluted with 12 mL of a hexane-dichloromethane mixed eluent with a volume ratio of 4:1. All eluent was collected and concentrated under reduced pressure to near dryness.

[0083] (7) The concentrate was diluted to volume with 1 mL of n-hexane and filtered through a 0.22 μm organic filter membrane to obtain the test solution;

[0084] (8) The test solution was injected into a gas chromatograph-tandem mass spectrometer. The gas chromatograph used a DB-5MS capillary column with the following column temperature program: initial temperature 40℃, hold for 1 min, increase to 200℃ at 20℃ / min, then increase to 300℃ at 5℃ / min and hold for 10 min. The mass spectrometer used an electron impact ion source with an ion source temperature of 280℃, high-purity helium as the carrier gas with a flow rate of 1.0 mL / min, an injection port temperature of 290℃, and an injection volume of 1.0 μL. Polycyclic aromatic hydrocarbons and polychlorinated biphenyls were detected using multiple reaction monitoring mode.

[0085] (9) Deuterated polycyclic aromatic hydrocarbons, 13 A standard curve was established using C-labeled polychlorinated biphenyls as internal standards, and the contents of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in the samples were calculated.

[0086] Example 2

[0087] Samples of Liubao tea produced using modern fermentation technology in Wanxiu District, Wuzhou, Guangxi were selected. All operations were completed according to the process parameters in Example 1. After purification, the test solution was colorless and transparent, free from interference from pigments and macromolecular impurities. Baseline separation was achieved for all 16 PAHs and 7 PCBs chromatographic peaks, and the mass spectrometry response signal was stable.

[0088] Example 3

[0089] Three-year-aged Liubao tea samples from Tengxian County, Wuzhou, Guangxi were selected. Pretreatment and detection were completed according to the process parameters of Example 1. There was no loss of target pollutants during extraction. The internal standard and target analytes responded synchronously. The quantitative results showed good repeatability and no obvious deviation.

[0090] Example 4

[0091] Wild Liubao tea samples from Mengshan County, Wuzhou, Guangxi were selected and tested according to the process parameters in Example 1. The method can accurately detect trace naphthalene contaminants in the samples, while PCBs contaminants were not detected. The sensitivity meets the requirements for the detection of trace contaminants.

[0092] Example 5

[0093] A sample of lightly fermented Liubao tea from Cenxi City, Wuzhou, Guangxi Province was selected. All operations were completed according to the process parameters of Example 1. The pretreatment process was smooth, the purification effect was excellent, the instrument detection showed no matrix interference peaks, and the quantitative results were accurate and reliable.

[0094] Example 6

[0095] Five-year-aged Liubao tea samples were selected from Dieshan District, Wuzhou, Guangxi, and the tests were performed according to the process parameters in Example 1. The test results of parallel samples from different batches showed high consistency, and the stability and reproducibility of the method met the requirements for batch testing.

[0096] Comparative Example 1

[0097] The only difference from Example 1 is that the extractant used is a single n-hexane solvent; all other process parameters and operating steps are exactly the same. Single n-hexane cannot penetrate the compact matrix of Liubao tea, cannot release bound PAHs and PCBs, resulting in extremely low extraction efficiency, severe matrix interference, and an inability to achieve accurate quantification.

[0098] Comparative Example 2

[0099] The only difference from Example 1 is that acetone is used as the single solvent for extraction, while the other process parameters and operating steps are exactly the same. Using acetone alone will dissolve a large amount of polar impurities, drastically increasing purification pressure, causing significant matrix effects, severe peak overlap, and extremely large deviations in detection results.

[0100] Comparative Example 3

[0101] The only difference from Example 1 is that the ultrasonic extraction time was set to 10 minutes; all other process parameters and operating steps were exactly the same. If the ultrasonic time is too short, cell disruption will be insufficient, resulting in inadequate dissolution of contaminants, weak peak response of the target analyte, and a risk of missed detection.

[0102] Comparative Example 4

[0103] The only difference from Example 1 is that the ultrasonic extraction time was set to 40 min; all other process parameters and operating steps were exactly the same. Excessive ultrasonic time leads to the volatilization and decomposition of low-boiling-point PAHs due to heat generation, while also causing excessive leaching of impurities and a significant decrease in the signal-to-noise ratio.

[0104] Comparative Example 5

[0105] The only difference from Example 1 is that the solid-phase extraction column uses a single Florisil packing material; all other process parameters and operating steps are exactly the same. A single packing material cannot remove tea pigments and large molecular protein impurities, resulting in a dark brown sample solution and extremely high background noise in the mass spectrometer.

[0106] Comparative Example 6

[0107] The only difference from Example 1 is that the solid-phase extraction column uses a single activated carbon packing material; all other process parameters and operating steps are exactly the same. Excessive activated carbon adsorption of the target pollutants led to a significant decrease in the recovery rates of PAHs and PCBs, resulting in severely underestimated detection results.

[0108] Comparative Example 7

[0109] The only difference from Example 1 is that the eluent volume is set to 5 mL; all other process parameters and operating steps are exactly the same. Insufficient eluent will result in incomplete elution of the target contaminant, leaving a large amount remaining on the solid-phase extraction column and distorting the detection results.

[0110] Comparative Example 8

[0111] The only difference from Example 1 is that the eluent volume is set to 20 mL; all other process parameters and operating steps are exactly the same. Excessive eluent usage leads to simultaneous elution of impurities adsorbed by the packing material, resulting in purification failure and the inability to eliminate matrix interference.

[0112] Comparative Example 9

[0113] The only difference from Example 1 is that gas chromatography-mass spectrometry (GC-MS) full scan mode was used for detection; all other process parameters and operating procedures were exactly the same. Full scan mode collects a large number of matrix ions, resulting in high background noise and insufficient sensitivity, which cannot meet the requirements for trace detection at the μg / kg level.

[0114] Comparative Example 10

[0115] The only difference from Example 1 is that the external standard method is used for quantification; all other process parameters and operating steps are exactly the same. Compared with the internal standard method used in this invention, the external standard method cannot effectively compensate for the matrix effect caused by the complex matrix of Liubao tea and the loss of target substances during the pretreatment process. As a result, the precision and accuracy of the results in the detection of samples from different batches and different origins are significantly lower than those of the method of this invention.

[0116] Single-factor screening experiment for key process parameters

[0117] Using the average recovery rate of the target analyte, signal-to-noise ratio (S / N), and chromatographic resolution as evaluation indicators, single-factor screening of core process parameters was carried out. The data were objective, true, and without duplicate values, and the conclusions clearly demonstrated the necessity of parameter selection.

[0118] Experiment 1: Screening of extraction solvent volume ratio (n-hexane: acetone).

[0119] Following the operating procedure of Example 1, only the volume ratio of the hexane-acetone composite extractant was changed, setting five gradients: 2:1, 2.5:1, 3:1, 3.5:1, and 4:1, while keeping the other parameters fixed. The average recovery rate and signal-to-noise ratio of the target analyte were measured to evaluate the effect of different ratios on the extraction effect. The results are shown in Table 1.

[0120]

[0121] Table 1 shows that when the volume ratio is <2.5:1, the acetone content is too high, leading to the dissolution of a large amount of polar tea polyphenols and tea polysaccharides, a significant increase in matrix interference, and a decrease in the signal-to-noise ratio. When the volume ratio is >3.5:1, the acetone content is insufficient, failing to break the binding bonds between pollutants and the matrix, resulting in incomplete extraction of bound pollutants and a significant decrease in recovery rate. The optimal range is 2.5:1-3.5:1, with 3:1 being the best.

[0122] Experiment 2: Screening of ultrasonic extraction time.

[0123] Following the operating procedure of Example 1, only the ultrasonic extraction time was changed, setting five gradients of 10 min, 20 min, 25 min, 30 min, and 40 min, while keeping other parameters unchanged. The average recovery rate and signal-to-noise ratio of the target analyte were detected to evaluate the effect of ultrasonic time on the dissolution efficiency of pollutants. The detection results are shown in Table 2.

[0124]

[0125] Table 2 shows that when the time is less than 20 min, the ultrasonic cavitation effect is insufficient, the tea cell disruption is incomplete, and the amount of pollutants dissolved is low; when the time is greater than 30 min, the ultrasonic heat generation leads to the volatilization and decomposition of low-boiling-point PAHs, while the amount of impurities dissolved increases, and the detection accuracy decreases. The optimal range is 20-30 min, with 25 min being the best.

[0126] Experiment 3: Screening of mixed filler mass ratio (Florida silica: activated carbon).

[0127] Referring to the operating process of Example 1, only the mass ratio of Florisil to activated carbon was changed, setting five gradients: 3:1, 3.5:1, 4:1, 4.5:1, and 5:1, while keeping other parameters unchanged. The average recovery rate and signal-to-noise ratio of the target analyte were measured to evaluate the impact of the packing ratio on the purification effect. The test results are shown in Table 3.

[0128]

[0129] Table 3 shows that when the activated carbon ratio is greater than 4.5:1, excessive activated carbon non-specifically adsorbs the target pollutants, resulting in a decreased recovery rate; when the activated carbon ratio is less than 3.5:1, pigments and macromolecular impurities are not completely removed, matrix interference cannot be eliminated, and the signal-to-noise ratio decreases. The optimal range is 3.5:1-4.5:1, with 4:1 being the best.

[0130] Experiment 4: Elution agent volume screening.

[0131] Following the operating procedure of Example 1, only the volume of the hexane-dichloromethane mixed eluent was changed, setting five gradients of 5 mL, 10 mL, 12 mL, 15 mL, and 20 mL, while keeping other parameters unchanged. The average recovery rate and signal-to-noise ratio of the target analyte were measured to evaluate the effect of eluent dosage on the enrichment effect of the target analyte. The test results are shown in Table 4.

[0132]

[0133] Table 4 shows that when the volume is <10 mL, the target pollutant is not completely eluted and remains on the solid-phase extraction column, resulting in lower results. When the volume is >15 mL, impurities are eluted simultaneously, the purification effect is lost, and the signal-to-noise ratio decreases. The optimal range is 10-15 mL, with 12 mL being the best.

[0134] Experiment 5: Screening of heating rate for gas chromatography column.

[0135] Following the operating procedure of Example 1, only the heating rate of the gas chromatography column was changed, setting five gradients of 3℃ / min, 4℃ / min, 5℃ / min, 6℃ / min, and 7℃ / min, while keeping the other chromatographic parameters fixed. The chromatographic resolution of the target analyte was detected, and the effect of the heating rate on the separation effect was evaluated. The detection results are shown in Table 5.

[0136]

[0137] Table 5 shows that when the chromatographic rate is <4℃ / min, the peak broadening is severe and the response value decreases; when the rate is >6℃ / min, the peak separation between PAHs and PCBs decreases, and peak overlap occurs, making accurate quantification impossible. The optimal range is 4-6℃ / min, with 5℃ / min being the best.

[0138] Experiment 6: Screening of mass spectrometry ion source temperature.

[0139] Following the operating procedure of Example 1, only the temperature of the mass spectrometry electron bombardment ion source was changed, setting five gradients of 250℃, 270℃, 280℃, 290℃, and 300℃, while keeping the other mass spectrometry parameters fixed. The signal-to-noise ratio and average recovery rate of the target analyte were detected, and the effect of the ion source temperature on the accuracy and sensitivity of the method was evaluated. The detection results are shown in Table 6.

[0140]

[0141] As shown in Table 6, when the temperature is <270℃, the target analyte response value is low, resulting in unsatisfactory signal-to-noise ratio and average recovery rate. When the temperature is >290℃, the target analyte may undergo excessive pyrolysis, weakening the characteristic ion signal, which also leads to a decrease in detection performance. The optimal range is 270-290℃, with 280℃ being the best.

[0142] Experiment 7: Carrier gas flow rate screening.

[0143] Following the operating procedure of Example 1, only the flow rate of the gas chromatographic carrier gas (high-purity helium) was changed, setting five gradients of 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, and 1.2 mL / min, while keeping the other chromatographic parameters fixed. The chromatographic resolution and signal-to-noise ratio of the target analyte were detected, and the effect of the carrier gas flow rate on the separation and response was evaluated. The detection results are shown in Table 7.

[0144]

[0145] Table 7 shows that when the flow rate is <0.9 mL / min, the chromatographic peak broadens and the detection time increases; when the flow rate is >1.1 mL / min, the retention time of the target analyte shortens, the resolution decreases, and the signal-to-noise ratio decreases. The optimal range is 0.9-1.1 mL / min, with the best value being 1.0 mL / min.

[0146] Experiment 8: Sample volume screening.

[0147] Following the operating procedure of Example 1, only the injection volume was changed, setting five gradients of 0.5 μL, 0.8 μL, 1.0 μL, 1.2 μL, and 2.0 μL, while keeping the other instrument parameters fixed. The signal-to-noise ratio and peak symmetry of the target analyte were detected, and the influence of the injection volume on the detection effect was evaluated. The detection results are shown in Table 8.

[0148]

[0149] Table 8 shows that when the injection volume is <0.8 μL, the target analyte response value is insufficient and the signal-to-noise ratio is low; when the injection volume is >1.2 μL, the column is overloaded, the peak shape is distorted, and the quantitative accuracy decreases. The optimal range is 0.8-1.2 μL, with 1.0 μL being the best.

[0150] Performance index testing:

[0151] This experiment was conducted in accordance with GB5009.265-2021 "National Food Safety Standard - Determination of Polycyclic Aromatic Hydrocarbons in Food" and GB5009.190-2014 "National Food Safety Standard - Determination of Indicative Polychlorinated Biphenyls in Food". All performance indicators of Examples 1-6 and Comparative Examples 1-10 were tested. The test indicators included the average recovery rate of PAHs, the average recovery rate of PCBs, precision (RSD, n=6), and signal-to-noise ratio (S / N).

[0152] I. Testing and experimental conditions

[0153] 1. Test samples: Examples 1-6 are Liubao teas from different production areas / processes / aging years in Wuzhou, Guangxi. Comparative examples 1-10 are traditional fermented Liubao teas from Cangwu County, Wuzhou, Guangxi, which are the same as in Example 1 (ensuring consistency of matrix, only changing the process / parameters).

[0154] 2. Number of replicate tests: All samples were measured in parallel 6 times (n=6), and the precision was the relative standard deviation of the 6 results;

[0155] 3. Recovery rate determination: A low-concentration spiking mode was adopted (the spiking amount was 1 / 10 of the limit value in GB2762-2022), which is consistent with the actual trace detection scenario.

[0156] II. Performance Index Test Results Table of Examples 1-6 and Comparative Examples 1-10

[0157]

[0158] III. Analysis of Test Results and Conclusions

[0159] (I) Analysis of test results in Examples 1-6

[0160] 1. Recovery rate and precision: The average recovery rate of PAHs in Examples 1-6 was 88.47%-92.36%, and the average recovery rate of PCBs was 89.23%-92.58%, both within the ideal recovery rate range of 85%-95% for food testing. The precision RSD of the six parallel determinations was 2.09%-2.67%, all less than 3%, indicating that the process parameters of the present invention have excellent adaptability to Liubao tea from different production areas, different processing techniques, and different aging years in Wuzhou, Guangxi. The extraction and purification effects are stable, the quantitative results have excellent repeatability, and there is no performance fluctuation caused by significant matrix differences.

[0161] 2. Signal-to-noise ratio: The signal-to-noise ratio of Examples 1-6 is 26.94-27.82, which is much higher than the S / N=3 requirement for trace detection.

[0162] (II) Analysis of the test results of Comparative Examples 1-10

[0163] Comparative Examples 1-10 all exhibited varying degrees of deterioration in performance indicators due to the use of a single reagent, deviations from the optimal parameter range, and unreasonable packing materials / detection modes / quantitative methods, and none of them met the national standards for food testing. The specific defects are as follows:

[0164] 1. Single extraction solvent (Comparative Examples 1-2): Single hexane cannot release bound pollutants, and single acetone dissolves a large amount of polar impurities. Both lead to a significant decrease in recovery rate (PAHs<71%, PCBs<73%) and RSD>8.86%. This verifies that the 3:1 hexane-acetone composite extractant is a necessary condition for the efficient extraction of the two types of pollutants. The composite solvent with complementary polarities can balance the extraction selectivity and extraction efficiency.

[0165] 2. Ultrasonic time deviates from the optimal range (Comparative Examples 3-4): Ultrasonic time of 10 min is insufficient for cavitation effect, resulting in low pollutant dissolution. Ultrasonic time of 40 min generates heat, leading to the volatilization of low-boiling-point PAHs and excessive dissolution of impurities. The recovery rates of both are <69% and RSD>8.23%, which verifies the necessity of ultrasonic time of 20-30 min (optimal 25 min), which can balance cell disruption efficiency and pollutant stability.

[0166] 3. Solid phase extraction using a single packing material (Comparative Examples 5-6): Florisil alone cannot remove large molecular impurities such as tea pigments, and activated carbon alone does not specifically adsorb the target pollutants. Among them, Comparative Example 6 had the lowest recovery rate of less than 50%, which verified that the synergistic purification effect of the 4:1 Florisil-activated carbon mixed packing material is the core to eliminating the interference of the complex matrix of Liubao tea. Both packing materials are indispensable.

[0167] 4. Eluent volume deviates from the optimal range (Comparative Examples 7-8): 5 mL of eluent resulted in incomplete elution of the target analyte, while 20 mL resulted in simultaneous elution of impurities. The recovery rates of both were <79% and RSD>8.52%, which verified that an eluent volume of 10-15 mL (optimal 12 mL) can ensure complete elution of the target analyte without interference from impurities, and is a key parameter for enrichment and purification.

[0168] 5. Inappropriate detection mode (Comparative Example 9): The GC-MS full scan mode collected a large number of matrix ions, and the signal-to-noise ratio dropped sharply to 8.72 (the lowest among all samples).

[0169] 6. Inappropriate quantitative method (Comparative Example 10): Decreased recovery rate and RSD as high as 9.31%, verifying the presence of deuterated PAHs+. 13 The C-labeled PCBs stable isotope internal standard method can systematically eliminate systematic errors, which is a necessary condition to ensure the accuracy of quantitative results.

Claims

1. An analytical method for simultaneously detecting polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in Liubao tea, characterized in that, Includes the following steps: (1) Sample pretreatment: The Liubao tea sample was pulverized at low temperature and then sieved. The sample was then accurately weighed and placed in a centrifuge container. (2) Composite solvent extraction: Add n-hexane-acetone composite extractant to the sample, perform ultrasonic-assisted extraction, centrifuge after extraction, and collect the upper organic phase; (3) Preliminary concentration: The organic phase is concentrated under reduced pressure to near dryness to obtain crude extract of the sample; (4) Activation of mixed solid phase extraction column: Florisil-activated carbon mixed solid phase extraction column was used and activated with n-hexane and acetone in sequence; (5) Sample loading and purification: The crude extract is dissolved in n-hexane and then loaded onto the packing material. The flow rate is controlled so that the target substance is adsorbed onto the packing material and impurities are removed. (6) Elution and enrichment: The solid phase extraction column was eluted with a mixture of n-hexane and dichloromethane as eluent, the eluent was collected and concentrated to near dryness under reduced pressure; (7) Volume adjustment and filtration: The solution is adjusted to volume with n-hexane and then filtered through an organic filter membrane to obtain the test solution; (8) Instrument detection: The test solution is injected into the gas chromatograph-tandem mass spectrometer, and polycyclic aromatic hydrocarbons and polychlorinated biphenyls are detected simultaneously using the multiple reaction monitoring mode; (9) Internal standard quantification: A standard curve was established using stable isotope internal standards to calculate the content of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in the sample.

2. The analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea according to claim 1, characterized in that, The volume ratio of the hexane-acetone composite extractant in step (2) is 2.5:1-3.5:1, the ultrasonic extraction temperature is 25-35℃, and the ultrasonic time is 20-30min.

3. The analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea according to claim 1, characterized in that, In step (2), the centrifugation speed is 7000-9000 r / min and the centrifugation time is 4-6 min.

4. The analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea according to claim 1, characterized in that, In step (4), the mass ratio of Florisil to activated carbon in the mixed solid-phase extraction column is 3.5:1-4.5:1, and the packing material specification is 500mg / 6mL.

5. The analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea according to claim 1, characterized in that, The volume ratio of hexane to dichloromethane eluent in step (6) is 3.5:1-4.5:1, and the total amount of eluent used is 10-15 mL.

6. The analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea according to claim 1, characterized in that, In step (7), the pore size of the organic filter membrane is 0.22 μm and the fixed volume is 1 mL.

7. The analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea according to claim 1, characterized in that, In step (8), the gas chromatography uses a DB-5MS capillary column with the following column temperature program: initial temperature 40℃ held for 1 min, temperature increased to 200℃ at 20℃ / min, and then temperature increased to 300℃ at 4-6℃ / min held for 10 min.

8. The analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea according to claim 1, characterized in that, In step (8), the mass spectrometer uses an electron impact ion source with an ion source temperature of 270-290℃, and the carrier gas is high-purity helium with a carrier gas flow rate of 0.9-1.1 mL / min.

9. The analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea according to claim 1, characterized in that, In step (8), the injection port temperature is 280-300℃, the injection volume is 0.8-1.2μL, and the splitless injection mode is used.

10. The analytical method for simultaneously detecting polycyclic aromatic hydrocarbons and polychlorinated biphenyls in Liubao tea according to claim 1, characterized in that, The internal standard mentioned in step (9) is a mixture of deuterated polycyclic aromatic hydrocarbon standards and... 13 C-labeled polychlorinated biphenyl mixed standards.

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