UPC for detecting polycyclic aromatic hydrocarbons in milk 2 - rapid analysis method by triple quadrupole tandem mass spectrometry

Abstract

A UPC for detecting polycyclic aromatic hydrocarbons in milk 2 – A rapid analysis method using triple quadrupole mass spectrometry (TQMS) involves ultrasonic-assisted extraction of polycyclic aromatic hydrocarbons (PAHs) from milk samples, followed by purification and concentration of the extract, and then ultra-high performance chromatography-triple quadrupole mass spectrometry (UPCMS). 2 The concentrated sample was analyzed using a pre-prepared linear standard curve for polycyclic aromatic hydrocarbons (PAHs) to obtain the content of 17 PAHs in the milk. This invention can rapidly detect 17 PAHs in milk and has the advantages of good separation effect, short detection time, and high detection sensitivity.

Description

Technical Field

[0001] This invention relates to a technology in the field of food safety, specifically an ultra-high performance convergence chromatography (UPC) method for analyzing 17 polycyclic aromatic hydrocarbons in milk. 2 – A rapid analysis method using triple quadrupole tandem mass spectrometry. Background Technology

[0002] Current quantitative analyses of polycyclic aromatic hydrocarbons (PAHs) commonly employ chromatographic, spectroscopic, and immunological methods. Among these, HPLC-FLD and GC-MS are the most widely used. These methods offer advantages such as high resolution, high sensitivity, and low detection limits, and can simultaneously analyze multiple different PAHs. However, they require longer analysis times and consume larger amounts of solvent. Ultra-high performance phase chromatography (UPC) is another method... 2 Using low-viscosity supercritical CO2 as the mobile phase, UPC allows for high-flow-rate analysis with shorter analysis times. Compared to traditional chromatography, UPC... 2 The analysis speed is significantly improved, the amount of solvent used is greatly reduced, and good separation results can be achieved. Summary of the Invention

[0003] To address the aforementioned shortcomings of existing technologies, this invention proposes a UPC method for detecting polycyclic aromatic hydrocarbons (PAHs) in milk. 2 – The triple quadrupole tandem mass spectrometry method is a rapid analysis method that can quickly detect 17 polycyclic aromatic hydrocarbons in milk. It has the advantages of good separation effect, short detection time and high detection sensitivity.

[0004] This invention is achieved through the following technical solution:

[0005] This invention relates to a UPC for detecting polycyclic aromatic hydrocarbons (PAHs) in milk. 2 – A rapid analysis method using triple quadrupole mass spectrometry (UPC) involves ultrasonic-assisted extraction of polycyclic aromatic hydrocarbons (PAHs) from milk samples, followed by purification and concentration of the extract, and then ultra-high performance chromatography-triple quadrupole mass spectrometry (UPC). 2 The concentrated sample was analyzed using the -QQQ-MS method, and the content of 17 polycyclic aromatic hydrocarbons in the milk was obtained by referring to the pre-prepared linear standard curve of polycyclic aromatic hydrocarbons.

[0006] The aforementioned ultra-high performance phase chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) refers to the separation of compounds using UHPLC followed by quantitative analysis using triple quadrupole mass spectrometry (TQUITY-UPC). Specifically, it employs an ACQUITY UPC with dimensions of 3.0 mm × 100 mm and a diameter of 1.8 μm. 2The chromatographic column used was an HSS C18 SB column; mobile phase A was supercritical CO2, and mobile phase B was methanol; column temperature was 40℃; flow rate was 1.0–1.5 mL / min; system back pressure was 1600 psi; injection volume was 5.0 μL; ionization mode was atmospheric pressure chemical ionization (APCI+); scanning mode was single ion monitoring scan. This invention can achieve simultaneous separation and detection of 17 polycyclic aromatic hydrocarbons within 11 min.

[0007] The linear standard curve for the aforementioned polycyclic aromatic hydrocarbons (PAHs) was prepared as follows: Eight PAH standard solutions with concentrations of 1, 2, 5, 10, 20, 40, 60, and 100 μg / kg were prepared using n-hexane as the solvent, and quantification was performed using the external standard method. The concentration of PAHs in the standard solutions was plotted on the x-axis, and the curve was plotted using the UPC-based linear standard curve. 2 A linear standard curve was constructed using the response values ​​of PAHs after detection by QQQ-MS, i.e., the peak area, as the ordinate.

[0008] The 17 polycyclic aromatic hydrocarbons mentioned include: benzo[a]anthracene (BaA), (Chr), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), ind[c,d]pyrene (IP), dibenzo[a,h]anthracene (DahA), benzo[g,h,i]perylene (BghiP), cyclopentene[c,d]pyrene (CP), dibenzo[a,e]pyrene (DaeP), dibenzo[a,h]pyrene (DahP), dibenzo[a,i]pyrene (DaiP), dibenzo[a,l]pyrene (DalP), 5-methyl (5-MChr), benzo[c]fluorene (BcF), 9-fluorenone (9–FO), anthraquinone (ATQ).

[0009] The UPC mentioned 2 -QQQ-MS is used for the analysis of concentrated samples. Specific parameters are as follows:

[0010] Column: ACQUITY UPC 2 HSS C18 SB, 3.0mm×100mm, 1.8μm;

[0011] Column temperature: 40℃;

[0012] Mobile phase A is supercritical CO2, and mobile phase B is methanol; the flow rate is 1.0–1.5 mL / min.

[0013] System back pressure: 1600 psi;

[0014] Injection volume: 5 μL;

[0015] The gradient elution program was as follows: 0.0–0.5 min, mobile phase B volume fraction was 1%, flow rate 1.5 mL / min; 8.0–10.5 min, mobile phase B volume fraction increased from 10% to 20%, flow rate maintained at 1.5 mL / min; 11.0–11.1 min, mobile phase B volume fraction increased from 35% to 50%, flow rate decreased from 1.5 mL / min to 1.0 mL / min; 12.5–13.0 min, mobile phase B volume fraction decreased from 50% to 1%, flow rate maintained at 1.0 mL / min; at 13.5 min, flow rate increased to 1.5 mL / min.

[0016] Ionization mode: APCI+;

[0017] Capillary voltage: 2kV;

[0018] Degassing solvent flow rate: 1000 L / h;

[0019] Ion source temperature: 450℃;

[0020] Scanning method: Single ion monitoring (SIM);

[0021] Select ion monitoring: 181, 209, 217, 227, 229, 243, 253, 277, 279, 303.

[0022] Technical effect

[0023] This invention comprehensively solves the problems of long analysis time and high detection limits in existing technologies, which fail to meet the requirements of food testing. Specifically, addressing the issue that polycyclic aromatic hydrocarbons (PAHs) are composed of fused benzene rings and are not easily fragmented, this invention optimizes chromatographic conditions, including mobile phase composition, flow rate, column temperature, and back pressure, to achieve better separation and facilitate subsequent quantitative analysis. PAHs are mostly composed of fused aromatic rings, and the parent PAHs lack substituents, making them difficult to break down by mass spectrometry, resulting in low mass spectrometric response and difficulty in lowering the detection limit to meet the requirements of food analysis. Compared with existing technologies, this invention optimizes mass spectrometry conditions, employing an atmospheric pressure ionization (APCI) ion source more suitable for weakly polar compounds. Simultaneously, it optimizes capillary voltage, monitoring ions, ion source temperature, and compensation solvent, achieving rapid separation and detection of 17 PAHs with high resolution, good selectivity, and excellent separation effect. Meanwhile, by optimizing parameters such as mass spectrometry detection ions, capillary voltage, and ion source temperature, this invention achieves quantitative analysis. The detection limits for 17 target analytes are 0.24-1.33 μg / kg, and the quantitation limits are 0.79-4.39 μg / kg, demonstrating high sensitivity and meeting the requirements for quantitative analysis. Attached Figure Description

[0024] Figure 1 Here are the chromatograms of the 17 PAHs standards in Example 1;

[0025] In the diagram: 1 is 9-FO, 2 is ATQ, 3 is BcF, 4 is BaA, 5 is Chr, 6 is CP, 7 is 5-MChr, 8 is BkF, 9 is BbF, 10 is BaP, 11 is DahA, 12 is IP, 13 is BghiP, 14 is DalP, 15 is DaeP, 16 is DaiP, and 17 is DahP. Detailed Implementation

[0026] Example 1

[0027] This embodiment specifically includes the following steps:

[0028] The first step involved preparing linear standard curves for 17 PAHs. Specifically, this included preparing PAH standard solutions at eight concentration levels (1, 2, 5, 10, 20, 40, 60, and 100 μg / kg) using n-hexane as the solvent. The concentrations of PAHs in the standard solutions were plotted on the x-axis, and the curves were plotted on the y-axis using UPC. 2 A linear standard curve was constructed using the response values ​​of PAHs after detection by QQQ-MS, i.e., the peak area, as the ordinate. The method detection limit was determined by the concentration of the standard at a signal-to-noise ratio (S / N = 3) for each compound, and the method quantitation limit was determined by the concentration of the standard at a signal-to-noise ratio (S / N = 10) for each compound.

[0029] UPC used 2 The detection conditions for -QQQ-MS are as follows:

[0030] Column: ACQUITY UPC 2 HSS C18 SB, 3.0mm×100mm, 1.8μm;

[0031] Column temperature: 40℃;

[0032] Mobile phase A is supercritical CO2, and mobile phase B is methanol; the flow rate is 1.0–1.5 mL / min.

[0033] System back pressure: 1600 psi;

[0034] Injection volume: 5 μL;

[0035] The gradient elution program was as follows: 0.0–0.5 min, mobile phase B volume fraction was 1%, flow rate 1.5 mL / min; 8.0–10.5 min, mobile phase B volume fraction increased from 10% to 20%, flow rate maintained at 1.5 mL / min; 11.0–11.1 min, mobile phase B volume fraction increased from 35% to 50%, flow rate decreased from 1.5 mL / min to 1.0 mL / min; 12.5–13.0 min, mobile phase B volume fraction decreased from 50% to 1%, flow rate maintained at 1.0 mL / min; at 13.5 min, flow rate increased to 1.5 mL / min.

[0036] Ionization mode: APCI+;

[0037] Capillary voltage: 2kV;

[0038] Degassing solvent flow rate: 1000 L / h;

[0039] Ion source temperature: 450℃;

[0040] Scanning method: Single ion monitoring (SIM);

[0041] Selected ion monitoring values: 181, 209, 217, 227, 229, 243, 253, 277, 279, 303.

[0042] The second step involved extracting PAHs from milk using ultrasound-assisted extraction. The specific steps included: adding 10 mL of acetonitrile / acetone mixture (3:2, v / v) to 5 g of milk, vortexing for 30 seconds, sonicating for 5 minutes, and centrifuging at 4000 rpm for 5 minutes. The supernatant was collected in a centrifuge tube, and the extraction process was repeated twice. The extracts were combined and concentrated to 200-800 mg under a nitrogen stream at 20°C. Subsequently, 2 mL of acetonitrile / acetone mixture (3:2, v / v) was added to the concentrate, vortexed for 15 seconds, and centrifuged at 4000 rpm for 5 minutes. The supernatant was collected, and the extraction was repeated three times.

[0043] The third step is to purify and concentrate the extract, which includes the following steps:

[0044] 3.1) Activate the Welchrom C18E extraction column sequentially with 24 mL of methanol and 24 mL of acetonitrile;

[0045] 3.2) Add the solution to be purified to the extraction column, collect the eluent, and then rinse with 5 mL of acetonitrile / acetone (3:2, v / v), collect the eluent, concentrate the eluent to about 50 mg by nitrogen blowing, and add 1 mL of n-hexane.

[0046] 3.3) Activate the Welchrom Florisil PR extraction column sequentially with 10 mL of dichloromethane and 10 mL of n-hexane;

[0047] 3.4) Add the n-hexane solution from step 3.2) to the extraction column, discard the eluent, rinse with 5 mL of n-hexane first, then rinse the column with 5 mL of n-hexane / dichloromethane (19:1, v / v), discard the eluent;

[0048] 3.5) Elute with 5 mL of n-hexane / dichloromethane (1:2, v / v), collect the eluent and concentrate it to 1 mL by nitrogen blowing, add 500 μL of toluene and continue blowing to about 20 mg, then make up to 250 μL with n-hexane for analysis.

[0049] The fourth step involves purifying the sample using UPC. 2 After QQQ-MS analysis, the content of various PAHs in milk was obtained by referring to the standard curve prepared in the first step.

[0050] This embodiment further validates the data in the following way: In blank milk samples, at a spiked level of 10 μg / kg, the retention times, recoveries, limits of detection (LODs), and limits of quantitation (LOQs) for 17 PAHs are shown in Table 1. It can be seen that the LODs for the 17 target analytes in this embodiment are 0.24-1.33 μg / kg, and the LODs are 0.79-4.39 μg / kg. At a spiked level of 10 μg / kg, the recoveries of each target analyte are between 53.00% and 105.50%, meeting the analytical requirements for PAHs in milk.

[0051] Table 1. Retention time, recovery rate, and limit of detection for 17 PAHs

[0052]

[0053]

[0054] In this embodiment, the chromatograms of the 17 PAHs standards are as follows: Figure 1 As shown in the figure, this embodiment demonstrates good separation capability for the main target analytes, high response to the target substances, and no obvious baseline noise or drift phenomena were observed, making it suitable for accurate quantitative analysis.

[0055] The above-described specific implementations can be partially adjusted by those skilled in the art in different ways without departing from the principles and purpose of the present invention. The scope of protection of the present invention is defined by the claims and is not limited to the above-described specific implementations. All implementation schemes within the scope of the claims are bound by the present invention.

Claims

1. A UPC for detecting polycyclic aromatic hydrocarbons in milk 2 –A rapid analysis method for triple quadrupole tandem mass spectrometry, characterized by… By performing ultrasonic-assisted extraction on polycyclic aromatic hydrocarbons (PAHs) in the milk sample to be tested, followed by purification and concentration of the extract, and then using ultra-high performance phase chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) to detect the concentrated sample, the content of 17 PAHs in the milk sample was obtained by referring to the pre-prepared linear standard curve of PAHs. The ultrasonic-assisted extraction of polycyclic aromatic hydrocarbons (PAHs) in the milk sample to be tested specifically includes: adding 10 mL of acetonitrile / acetone mixture (3:2, v / v) to 5 g of milk, vortexing for 30 s, sonicating for 5 min, centrifuging at 4000 rpm for 5 min, collecting the supernatant in a centrifuge tube, and repeating the above extraction steps twice; combining the extracts, concentrating to 200-800 mg in a nitrogen stream at 20 ℃, adding 2 mL of acetonitrile / acetone mixture (3:2, v / v) to the concentrate, shaking for 15 s, centrifuging at 4000 rpm for 5 min, collecting the supernatant, and repeating the extraction three times; The purification and concentration of the extract specifically includes: 1) Activate the Welchrom C18E extraction column sequentially with 24 mL of methanol and 24 mL of acetonitrile; 2) Add the solution to be purified to the extraction column, collect the eluent, and then rinse with 5 mL of acetonitrile / acetone 3:2, v / v, collect the eluent, concentrate the eluent to about 50 mg by nitrogen blowing, and add 1 mL of n-hexane. 3) Activate the Welchrom Florisil PR extraction column sequentially with 10 mL of dichloromethane and 10 mL of n-hexane; 4) Add the n-hexane solution from step 2) to the extraction column, discard the eluent, rinse with 5 mL of n-hexane first, then rinse the column with 5 mL of n-hexane / dichloromethane 19:1, v / v, and discard the eluent. 5) Elute with 5 mL of n-hexane / dichloromethane 1:2, v / v, collect the eluent and concentrate it to 1 mL by nitrogen blowing, add 500 μL of toluene and continue blowing to about 20 mg, then make up to 250 μL with n-hexane for analysis; The 17 polycyclic aromatic hydrocarbons mentioned include: benzo[a]anthracene, benzo[b]fluoranthracene, benzo[k]fluoranthracene, benzo[a]pyrene, indo[c, d]pyrene, dibenzo[a, h]anthracene, benzo[g, h, i]perylene, cyclopentene[c, d]pyrene, dibenzo[a, e]pyrene, dibenzo[a, h]pyrene, dibenzo[a, i]pyrene, dibenzo[a, l]pyrene, 5-methyl[a], benzo[c]fluorene, 9-fluorenone, and anthraquinone; The linear standard curve for the aforementioned polycyclic aromatic hydrocarbons (PAHs) was prepared as follows: Eight PAH standard solutions with concentrations of 1, 2, 5, 10, 20, 40, 60, and 100 μg / kg were prepared using n-hexane as the solvent. Quantification was performed using the external standard method. The concentration of PAHs in the standard solutions was plotted on the x-axis, and the curve was plotted on the y-axis. 2 The response values ​​of PAHs after QQQ-MS detection, i.e. peak area, were used as the ordinate to create a linear standard curve; The aforementioned ultra-high performance phase chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) refers to the separation of compounds using UHPLC followed by quantitative analysis using triple quadrupole mass spectrometry (TQUITY-UPC). Specifically, it employs an ACQUITY UPC with dimensions of 3.0 mm × 100 mm and a diameter of 1.8 μm. 2 HSS C18 SB column; mobile phase A: supercritical CO2, mobile phase B: methanol; column temperature: 40 ℃; flow rate: 1.0–1.5 mL / min; system back pressure: 1600 psi; injection volume: 5.0 μL. The gradient elution program was as follows: 0.0–0.5 min, mobile phase B volume fraction was 1%, flow rate was 1.5 mL / min; 8.0–10.5 min, mobile phase B volume fraction increased from 10% to 20%, flow rate was maintained at 1.5 mL / min; 11.0–11.1 min, mobile phase B volume fraction increased from 35% to 50%, flow rate decreased from 1.5 mL / min to 1.0 mL / min; 12.5–13.0 min, mobile phase B volume fraction decreased from 50% to 1%, flow rate was maintained at 1.0 mL / min. Ionization mode: APCI+; Capillary voltage: 2 kV; Degassing solvent flow rate: 1000 L / h; Ion source temperature: 450 ℃; Scanning method: Single ion monitoring scan; Select ion monitoring: 181, 209, 217, 227, 229, 243, 253, 277, 279, 303.

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