Method for testing content of polycyclic aromatic hydrocarbon in Chinese herbal medicine

Abstract

The invention relates to the field of analysis and detection, in particular to a method for testing the content of polycyclic aromatic hydrocarbon in Chinese herbal medicine. The method comprises the following steps: ultrasonically extracting polycyclic aromatic hydrocarbon in a sample by adopting normal hexane-acetone, concentrating an extracting solution, fixing the volume, performing gas chromatography separation and mass spectrometry detection, and combining an internal standard method for quantitative analysis. A selected ion monitoring mode is used to improve the detection sensitivity and accuracy, and 2-fluorobiphenyl and 4, 4 '-terphenyl-d14 are used as substitutes to mix a standard solution; naphthalene-d8, acenaphthyl-d10, phenanthrene-d10, # imgabs0 #-d12 and perylene-d12 are used as internal standard substance mixed standard solutions, so that the stability and reliability of the analysis process are ensured, and an effective detection scheme is provided for quality control and safety evaluation of Chinese herbal medicines.

Description

Technical Field

[0001] The present invention relates to the field of analytical detection, and specifically, to a method for testing the content of polycyclic aromatic hydrocarbons in Chinese herbal medicines. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are strong carcinogenic pollutants composed of multiple benzene rings, and are widely present in industrial emissions, vehicle exhausts and combustion products in the environment. The International Agency for Research on Cancer has clearly classified PAHs such as benzo[a]pyrene as Group 1 carcinogens. Long-term exposure can cause diseases such as lung cancer and skin cancer, and cause long-term toxicity to the ecosystem. As the core carrier of traditional medicine, the safety of Chinese herbal medicines is directly related to public health. However, PAHs can be enriched in medicinal materials through ways such as environmental pollution during planting, such as polluted air and soil, incomplete combustion of fuels during the processing process, and contact with pollutants during storage and transportation. In particular, rhizome medicinal materials such as Angelica sinensis are more likely to adsorb and accumulate PAHs due to their high fat solubility and large surface area, resulting in potential health risks.

[0003] Currently, the detection of PAHs mostly relies on gas chromatography-mass spectrometry (GC-MS) technology. For example, Chinese Patent Application CN109358149A discloses a rapid quantitative detection method for polycyclic aromatic hydrocarbons in surface water. Its operation steps include PAHs extraction, high-temperature thermal desorption, cold focusing enrichment and GC-MS separation, and can quickly determine the content of ultra-trace PAHs in surface water. However, the detection of PAHs in the complex matrix of Chinese herbal medicines faces significant limitations: First, components such as volatile oils and polysaccharides in the medicinal materials are prone to interfering with the signals of target substances, reducing the detection specificity; Second, traditional pretreatment methods are time-consuming, require a large amount of solvents, and are difficult to efficiently separate PAHs; Third, the detection limits of low-concentration PAHs in complex matrices are relatively high, making it difficult to meet the strict limit requirements of institutions such as the Chinese Pharmacopoeia and the European Medicines Agency (EMA). In addition, existing research mostly focuses on food or environmental samples, lacking standardized detection methods for the matrix characteristics of Chinese herbal medicines, which restricts industry quality control and international trade compliance.

[0004] Therefore, there is an urgent need to develop an efficient, sensitive and specific detection method for polycyclic aromatic hydrocarbons in Chinese herbal medicines, providing a reliable quality control tool for Chinese herbal medicine production enterprises and at the same time providing a scientific basis for regulatory authorities to formulate international standards. Summary of the Invention

[0005] In order to solve the above technical problems, the first aspect of the present invention provides a method for testing the content of polycyclic aromatic hydrocarbons in Chinese herbal medicines, including:

[0006] S1. Grinding and sieving the sample to be tested, taking a sample and placing it in a centrifuge tube, adding a surrogate standard mixture solution, adding a first solvent, performing ultrasonic extraction treatment, and filtering to collect the supernatant;

[0007] S2. Rotate and dry the supernatant under a water bath, add a second solvent to mix and dissolve, add an internal standard mixed standard solution, then make up the volume, mix well, filter through an organic phase filter membrane, and collect the filtrate;

[0008] S3. Inject the filtrate into the instrument, and then detect the content of polycyclic aromatic hydrocarbons by GC-MS method.

[0009] As an implementable case, the polycyclic aromatic hydrocarbons to be measured in the present invention include: naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-c,d]pyrene, dibenzo[a,h]anthracene and benzo[g,h,i]perylene.

[0010] As an implementable case, the mesh number of the ground and sieved material is 50 - 100 meshes.

[0011] Further, the mesh number of the ground and sieved material is 50 meshes.

[0012] Selecting a 50-mesh sieve can control the particle size of the Chinese herbal medicine powder within a certain range, make the particle size more uniform, improve the extraction efficiency and stability. The uniform particle size can increase the contact area during extraction, facilitate the penetration of the solvent and the dissolution of components, and can avoid local solvent excess or deficiency caused by differences in particle size, thus ensuring the consistency of the extraction process. In addition, the sieved powder is more conducive to subsequent operations, can improve the extraction efficiency during ultrasonic extraction, reduce the risk of blockage during separation, and make the solution more uniform during volume determination, thereby improving the accuracy and repeatability of the measurement results.

[0013] As an implementable case, the standard substances in the surrogate mixed standard solution include 2-fluorobiphenyl and terphenyl-d 14 .

[0014] Further, the mass ratio of the 2-fluorobiphenyl and 4,4'-terphenyl-d 14 is (1 - 10):(1 - 10).

[0015] Furthermore, the mass ratio of the 2-fluorobiphenyl and 4,4'-terphenyl-d 14 is 1:1.

[0016] In the present invention, 2-fluorobiphenyl and 4,4'-terphenyl-d 14The surrogate mixed standard solution is mixed as a substitute mainly because its chemical properties are similar to those of the target polycyclic aromatic hydrocarbons, but it has unique mass spectrometry characteristics and can effectively monitor the extraction efficiency, instrument response, and method stability throughout the analysis process. By adding this mixture of two surrogates during sample pretreatment and determination, the surrogate mixed standard solution can accurately evaluate the recovery rate and detection accuracy of the target compounds in the actual sample, thus ensuring the reliability of the analysis results and the accuracy of the method. At the same time, they can also help identify and correct the deviations caused by matrix effects or instrument fluctuations, improving the accuracy and repeatability of the determination results.

[0017] As an implementable case, the first solvent includes n-hexane and acetone.

[0018] Furthermore, in the first solvent, the volume ratio of n-hexane to acetone is 1:1.

[0019] As an implementable case, the number of ultrasonic extraction times is 1 - 3 times, and the ultrasonic extraction time is 30 - 60 min.

[0020] Furthermore, the number of ultrasonic extraction times is 2 times, and the ultrasonic extraction time is 30 min.

[0021] As an implementable case, the temperature of the water bath is 35 - 50 °C.

[0022] Furthermore, the temperature of the water bath is 40 °C.

[0023] As an implementable case, the second solvent includes n-hexane and acetone.

[0024] Furthermore, in the second solvent, the volume ratio of n-hexane to acetone is 1:1.

[0025] As an implementable case, the standard substances in the internal standard mixed standard solution include naphthalene-d8, acenaphthene-d 10 , phenanthrene-d 10 , -d 12 and perylene-d 12 .

[0026] Furthermore, the mass ratio of naphthalene-d8, acenaphthene-d 10 , phenanthrene-d 10 , -d 12 and perylene-d 12 is (1 - 5):(1 - 5):(1 - 5):(1 - 5):(1 - 5).

[0027] Even further, the naphthalene-d8, acenaphthene-d 10 , phenanthrene-d10 , -d 12 and perylene-d 12 have a mass ratio of 1:1:1:1:1.

[0028] In the present invention, naphthalene-d8, acenaphthene-d 10 , phenanthrene-d 10 , -d 12 and perylene-d 12 are selected for compounding as the internal standard mixture solution. This is mainly because the chemical structures of these deuterated internal standards are similar to those of the target polycyclic aromatic hydrocarbons, but deuteration gives them unique mass-to-charge ratios (m / z) in mass spectrometry analysis, enabling effective differentiation of the signals of the target compounds and internal standards. By adding these internal standards with known concentrations to the sample, losses during sample pretreatment, variations in instrument response, and the influence of matrix effects can be corrected, thereby improving the accuracy and precision of quantitative analysis. In addition, the internal standards can also help monitor and optimize instrument performance, ensuring the stability and reliability of the entire analysis process, making the measurement results more accurate and reliable.

[0029] As an implementable case, the spiking amount of the internal standard mixture solution is 50 - 100 ng.

[0030] As an implementable case, in the GC-MS test, the temperature of the chromatographic column is 80 °C, then keep it warm for 1 - 3 min, increase the temperature at a rate of 15 - 20 °C / min to 180 °C, keep it warm for 5 - 10 min, and then increase the temperature at a rate of 5 - 10 °C / min to 290 °C, and keep it warm for 5 - 10 min.

[0031] As an implementable case, in the GC-MS test, the injection mode includes splitless injection.

[0032] As an implementable case, in the GC-MS test, the data acquisition mode includes SIM (Selected Ion Monitoring) mode.

[0033] Furthermore, in the GC-MS test:

[0034] Instrument name: Gas Chromatograph-Mass Spectrometer, Model: 8890 + 7000D;

[0035] Chromatographic column: Model HP-5, Specification: 30 m × 0.25 mm × 0.25 μm;

[0036] Column temperature: Initial temperature 80 °C, hold for 2 min, increase the temperature to 180 °C at a rate of 20 °C / min, hold for 5 min, then increase the temperature to 290 °C at a rate of 10 °C / min, and hold for 5 min;

[0037] Inlet temperature: 280°C;

[0038] Chromatography-mass spectrometry interface temperature: 280°C;

[0039] Ion source temperature: 230°C;

[0040] Carrier gas: helium, the flow rate of carrier gas is 1.0mL / min;

[0041] Injection volume: 1.0 μL;

[0042] Injection method: splitless injection;

[0043] Ionization method: EI;

[0044] Ionization energy: 70eV;

[0045] Solvent delay: 5min;

[0046] Data collection mode: SIM mode.

[0047] Beneficial Effects

[0048] (I) The present invention uses a gas chromatography-mass spectrometer in combination with the SIM mode, and performs quantitative analysis and process monitoring by means of an internal standard method and a substitute, which significantly improves the detection sensitivity and accuracy of polycyclic aromatic hydrocarbons in angelica. Experimental data show that the linear correlation coefficients of each compound are greater than 0.990, indicating that they have a good linear relationship in a wide concentration range, and can achieve accurate detection of low-content polycyclic aromatic hydrocarbons.

[0049] (ii) Through the optimization method, the present invention achieves low detection limit and low quantification limit for polycyclic aromatic hydrocarbons. The signal-to-noise ratio of all tested compounds is greater than 3, meeting the detection limit requirement; at a spiked amount of 50 ng, the signal-to-noise ratio is greater than 10, meeting the quantification limit requirement.

[0050] (III) The test method provided by the present invention has excellent precision. Measured by the repeatability relative standard deviation, the RSD of all target compounds does not exceed 15%, which helps to obtain comparable test results under different time, different operators or different equipment conditions.

[0051] (IV) The recovery rate of the test samples in the present invention ranges from 83.21% to 118.40%, which is in line with the standard recovery rate range of 70.0%-120.0%. This shows that the test method provided by the present invention can accurately recover and determine the polycyclic aromatic hydrocarbons in the angelica sample, ensuring the reliability of quantitative analysis, which is crucial for truly reflecting the actual content of polycyclic aromatic hydrocarbons in the sample.

[0052] (5) The operation process of the present invention is standardized. From sample pretreatment to instrument analysis and then to data acquisition and processing, each step has clear specifications and parameter settings, ensuring the repeatability and stability of the method. The process of ultrasonic extraction with n-hexane-acetone, gas chromatography separation, and mass spectrometry detection, combined with the use of the internal standard method and surrogates, effectively reduces the influence of matrix effects and operation errors, improves the reliability of the detection results, and provides a practical and effective detection means for the quality control and safety assessment of Angelica sinensis. Detailed implementation manners

[0053] Example 1

[0054] This example provides a method for testing the content of polycyclic aromatic hydrocarbons in Chinese herbal medicines, specifically as follows:

[0055] S1. Grind the Angelica sinensis sample to be tested, sieve it through a 50-mesh sieve, accurately weigh 1 g of the sample, place it in a 50-mL centrifuge tube, add 1 mL of the surrogate mixed standard solution, and then add 30 mL of n-hexane-acetone (1:1, v / v). Perform ultrasonic extraction 2 times, 30 min each time, and filter to collect the supernatant; the standard substances in the surrogate mixed standard solution (solvent: n-hexane-acetone (1:1, v / v), mass concentration of solute: 50 ng) are 2-fluorobiphenyl and 4,4'-terphenyl-d 14 ; the mass ratio of 2-fluorobiphenyl and 4,4'-terphenyl-d 14 is 1:1;

[0056] S2. Concentrate the supernatant by rotary evaporation in a 40 °C water bath, add 1 mL of n-hexane-acetone (1:1, v / v) to dissolve, add the internal standard mixed standard solution, and then add n-hexane-acetone (1:1, v / v) to make the volume up to 5 mL. Mix well, filter through an organic phase filter membrane, and collect the filtrate; the standard substances in the internal standard mixed standard solution are naphthalene-d8, acenaphthene-d 10 , phenanthrene-d 10 , -d 12 and perylene-d 12 ; the mass ratio of naphthalene-d8, acenaphthene-d 10 , phenanthrene-d 10 , -d 12 and perylene-d 12 is 1:1:1:1:1.

[0057] S3. Inject the filtrate into the instrument and then perform GC-MS content detection for the content of polycyclic aromatic hydrocarbons.

[0058] The polycyclic aromatic hydrocarbons to be tested in this example are: naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, Benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-c,d]pyrene, dibenzo[a,h]anthracene and benzo[g,h,i]perylene, the information of the standard substance of the analyte (i.e., the blank control sample of the analyte) and the mixed standard solution of the internal standard, and the mixed standard solution of the surrogate is shown in Table 1.

[0059] Table 1

[0060]

[0061]

[0062] In the GC-MS test described above:

[0063] Instrument name: Gas chromatography-mass spectrometry, Model: 8890 + 7000D.

[0064] Chromatographic column: Model HP-5, Specification: 30m × 0.25mm × 0.25μm;

[0065] Column temperature: Initial temperature 80°C, hold for 2 min, increase to 180°C at 20°C / min, hold for 5 min, then increase to 290°C at 10°C / min, hold for 5 min;

[0066] Injector temperature: 280°C;

[0067] Chromatography-mass spectrometry interface temperature: 280°C;

[0068] Ion source temperature: 230°C;

[0069] Carrier gas: Helium, carrier gas flow rate is 1.0 mL / min;

[0070] Injection volume: 1.0 μL;

[0071] Injection mode: Splitless injection;

[0072] Ionization mode: EI;

[0073] Ionization energy: 70 eV;

[0074] Solvent delay: 5 min;

[0075] Data acquisition mode: SIM mode, and the working setting parameters of the SIM mode are shown in Table 2.

[0076] Table 2

[0077]

[0078] 1. Linear relationship test

[0079] Construct linear equations with the injection concentrations set as: 5 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, 100 μg / L, 200 μg / L, and test the response values at each concentration. The experimental test results are shown in Table 3.

[0080] Table 3

[0081]

[0082]

[0083] The experimental results in Table 3 show that in this experiment, different injection concentrations were used, and the linear R was all > 0.99, indicating a significant linear relationship between the concentration and the response value, being stable and having good repeatability within the tested concentration range, and can be used for accurately quantifying the content of polycyclic aromatic hydrocarbon compounds in Angelica sinensis samples.

[0084] 2. Detection limit test

[0085] Test the signal-to-noise ratio and detection limit of 10 groups of filtrates of Angelica sinensis samples. The experimental results are shown in Tables 4 - 5.

[0086] Table 4

[0087]

[0088]

[0089] Table 5

[0090]

[0091]

[0092] It can be seen from the experimental results in Tables 4 - 5 that in this experiment, 5 μL of the reference solution was added to the blank matrix and measured in parallel 10 times, and the signal-to-noise ratio was all > 3; indicating that this method can reliably detect the target polycyclic aromatic hydrocarbon compounds, and has good anti-interference ability and reproducibility, both meeting the requirements of the pharmacopoeia.

[0093] 3. Quantitation limit detection

[0094] Detect the signal-to-noise ratio of the components in 10 groups of filtrates of Angelica sinensis samples. The experimental results are shown in Tables 6 - 7.

[0095] Table 6

[0096]

[0097]

[0098]

[0099] Table 7

[0100]

[0101]

[0102] In this experiment, 10 μL of the reference substance solution was added to the blank matrix, and parallel determinations were carried out 10 times. The signal-to-noise ratio was > 10, indicating that this method can accurately quantify the target compound, and the precision, anti-interference ability, and robustness all meet the requirements of the pharmacopoeia.

[0103] 4. Precision confirmation

[0104] The determined concentrations of the spiked samples of the filtrate components of 6 groups of Angelica sinensis samples were detected. The experimental results are shown in Tables 8 and 9.

[0105] Table 8

[0106]

[0107]

[0108] Table 9

[0109]

[0110]

[0111] In this experiment, 10 μL of the reference substance solution was added to the blank matrix, and parallel determinations were carried out 6 times. The relative standard deviation of repeatability was ≤ 15%.

[0112] 5. Quantitative determination

[0113] Six groups of filtrates of Angelica sinensis samples were taken, the reference substance was added, and vortex mixing was carried out. The maximum recovery rate and minimum recovery rate of the spiked samples of each component were detected. The experimental results are shown in Tables 10 - 11.

[0114] Table 10

[0115]

[0116]

[0117] Table 11

[0118]

[0119]

[0120] In this experiment, 10 μL of the reference substance solution was added during the pretreatment process of the blank matrix, and parallel determinations were carried out 6 times. The recovery rate range of the target analyte was 83.21% to 118.40%, and the standard recovery rate range was 70.0% - 120.0%, indicating that this test method can efficiently recover polycyclic aromatic hydrocarbon compounds.

Claims

1. A method for testing the content of polycyclic aromatic hydrocarbons in Chinese herbal medicines, characterized in that, Including: S1. Grind and sieve the sample to be tested, take a sample and place it in a centrifuge tube, add a surrogate standard solution, add a first solvent, perform ultrasonic extraction treatment, and filter to collect the supernatant; S2. Rotavaporize the supernatant under a water bath, add a second solvent to mix and dissolve, add an internal standard solution, then make up the volume, mix well, filter through an organic phase filter membrane, and collect the filtrate; S3. Inject the filtrate into the instrument, and then detect the content of polycyclic aromatic hydrocarbons by GC-MS.

2. The test method for the content of polycyclic aromatic hydrocarbons in Chinese herbal medicines according to claim 1, characterized in that, The mesh number of the sieve for the grinding and sieving is 50 - 100 meshes.

3. The method for testing the polycyclic aromatic hydrocarbon content in Chinese herbal medicines according to claim 1, characterized in that The reference substances in the alternative mixed standard solution include 2-fluorobiphenyl and 4,4'-terphenyl-d 14 .

4. The method for testing the polycyclic aromatic hydrocarbon content in Chinese herbal medicines according to claim 3, wherein, The 2-fluorobiphenyl and terphenyl-d described above 14 have a mass ratio of (1 - 10):(1 - 10).

5. The method for testing the polycyclic aromatic hydrocarbon content in Chinese herbal medicines according to claim 1, characterized in that, The first solvent includes n-hexane and acetone.

6. The method for testing the polycyclic aromatic hydrocarbon content in Chinese herbal medicines according to claim 1, characterized in that, The number of times of ultrasonic extraction is 1 - 3 times, and the time of ultrasonic extraction is 30 - 60 min.

7. The method for testing the polycyclic aromatic hydrocarbon content in Chinese herbal medicines according to claim 1, characterized in that, The standard substances in the internal standard mixed standard solution include naphthalene-d8, acenaphthene-d 10 , phenanthrene-d 10 , -d 12 and perylene-d 12 .

8. The test method for the content of polycyclic aromatic hydrocarbons in Chinese herbal medicines according to claim 7, characterized in that, The naphthalene-d8, acenaphthene-d 10 , phenanthrene-d 10 , -d 12 and perylene-d 12 have a mass ratio of (1 - 5) : (1 - 5) : (1 - 5) : (1 - 5) : (1 - 5).

9. The method for testing the polycyclic aromatic hydrocarbon content in Chinese herbal medicines according to claim 1, wherein In the GC-MS test, the temperature of the chromatographic column is 80 °C, then keep warm for 1 - 3 min, increase the temperature to 180 °C at a rate of 15 - 20 °C / min, keep warm for 5 - 10 min, increase the temperature to 290 °C at a rate of 5 - 10 °C / min, and keep warm for 5 - 10 min.

10. The test method for the content of polycyclic aromatic hydrocarbons in Chinese herbal medicines according to claim 1, characterized in that, In the GC-MS test, the data acquisition mode includes the SIM mode.

Images