Method for improving sensitivity of GC-MS method for determining organic fluorine in traditional Chinese medicinal materials

Abstract

The invention discloses a method for improving sensitivity of a GC-MS (gas chromatography-mass spectrometry) method for measuring organic fluorine in traditional Chinese medicinal materials. High-temperature headspace heating is combined with headspace treatment or conventional headspace heating temperature is combined with solid-phase microextraction treatment. By increasing the headspace heating treatment temperature (100-200 DEG C) of a sample, the volatilization separation speed and concentration of high-boiling-point and difficult-to-volatilize organic fluorine components are increased; or the organic fluorine is extracted and adsorbed at the conventional headspace heating temperature (lower than 100 DEG C) in combination with a solid-phase microextraction needle of a carbon-containing molecular sieve material, so that the separation and enrichment concentration of the organic fluorine is improved, and the sensitivity of determining the organic fluorine in the traditional Chinese medicinal materials by using a gas chromatography-mass spectrometry method is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of Chinese medicinal material detection, and in particular to a method for improving the sensitivity of GC-MS method in determining organic fluorine in Chinese medicinal materials. Background Art

[0002] There are many types of perfluorinated and polyfluorinated compounds, which are persistent organic pollutants with various potential toxicities. These compounds are difficult to detect due to their colorless and odorless nature, high boiling points, low volatility, poor solubility, and unique physical and chemical properties.

[0003] Currently, the main analytical methods for organic fluorine components reported in the literature include chemical methods, spectrophotometry, atomic absorption spectrometry, and chromatography-mass spectrometry. Chromatography-mass spectrometry, in particular, has become increasingly popular. The methods described in Reference 1 (T / DGAS037-2023, Detection of Organic Fluorides in Cordyceps Sinensis) suffer from cumbersome sample preparation, significant interference from impurities and extraction solvents, and low sensitivity, making them inadequate for rapid quality analysis and identification of Chinese medicinal materials such as Cordyceps.

[0004] However, the matrix of Chinese medicinal materials is complex, and the residual amount of organic fluorine components is mostly at trace levels. In order to obtain accurate and reliable detection data, in addition to highly sensitive detection instruments, efficient sample pretreatment methods are also required.

[0005] Headspace gas chromatography is often used to analyze and detect low-boiling-point, gaseous volatile components due to its ease of sample preparation, ability to eliminate interference from sample impurities and extraction solvents, and reduced instrument contamination. There are numerous perfluorinated and polyfluorinated organofluorine compounds, with significant variations in molecular structure, molecular weight, and physicochemical properties. They are insoluble in water and organic solvents, have high boiling points, and are difficult to volatilize. In particular, adsorbed and doped organofluorine components, such as perfluorotributylamine, perfluorotripentylamine, polyfluoroalkanes, and polyfluoroalkyl ethers, have been found in solid Chinese medicinal materials. These components have high molecular weights and boiling points ranging from 180°C to 300°C. Under conventional static headspace conditions with constant temperature equilibrium within 100°C, the concentration of organofluorine in the headspace gas is low, the chromatographic peak signal is weak, and they are susceptible to interference from other substances, making them difficult to accurately analyze and detect.

[0006] Chinese patent document CN116162217A discloses a method for detecting various perfluorinated and polyfluorinated alkyl compounds in traditional Chinese medicines based on solid-phase extraction adsorption materials. A uniform spherical covalent organic framework material was prepared by room-temperature synthesis. Experimental verification shows that the synthesis conditions are mild, the synthesis reagents are environmentally friendly, the material morphology is uniform and the pores are rich. It can be used as an efficient solid-phase extraction material in the detection and analysis of perfluorinated and polyfluorinated alkyl compounds in traditional Chinese medicines. However, the synthesis method of the adsorption material is complicated.

[0007] Therefore, it is urgent to establish a simple and sensitive method to detect organic fluorine components in traditional Chinese medicines to solve the problem of low sensitivity of organic fluorine detection in existing detection methods. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides a method for improving the sensitivity of GC-MS method for determining organic fluorine in traditional Chinese medicine. By increasing the temperature of headspace treatment or using solid phase microextraction needles containing carbon molecular sieve materials, the separation and enrichment concentration of organic fluorine is improved, thereby improving the sensitivity of gas chromatography-mass spectrometry method for determining organic fluorine in traditional Chinese medicine.

[0009] A method for improving the sensitivity of GC-MS determination of organic fluorine in traditional Chinese medicines, comprising the following steps: (1) Sample pretreatment: After the Chinese medicinal materials are crushed, they are placed in a headspace bottle, sealed with a cap, and subjected to headspace treatment at 100-200 °C to collect the headspace gas; alternatively, a solid phase microextraction needle containing carbon molecular sieve material is used to perform extraction at a temperature below 100 °C and collect the sample in the extraction needle; (2) The headspace gas or the sample in the extraction needle collected in step (1) is detected using a gas chromatography-mass spectrometer, and the type and content of organic fluorine in the Chinese medicinal materials are analyzed based on the standard curve of organic fluorine established in advance.

[0010] In this invention, for high-boiling-point, non-volatile organic fluorine components, high-temperature headspace heating combined with headspace treatment or conventional headspace heating combined with solid-phase microextraction (SPME) is used. By increasing the headspace heating temperature (100-200°C), the volatilization separation rate and concentration of high-boiling-point, non-volatile organic fluorine components are increased. Alternatively, conventional headspace heating (below 100°C) combined with solid-phase microextraction (SPME) needle extraction and adsorption of organic fluorine using carbon-containing molecular sieve materials can improve the separation and enrichment concentration of organic fluorine, thereby enhancing the sensitivity of gas chromatography-mass spectrometry for the determination of organic fluorine in traditional Chinese medicines.

[0011] Preferably, deoxygenation treatment is performed before the headspace treatment.

[0012] Further preferably, the deoxygenation step is to connect a nitrogen cylinder pressure reducing valve or a nitrogen blowdown device to the headspace bottle after the cap is sealed, and introduce nitrogen to replace the air in the headspace bottle.

[0013] For high-boiling-point organic fluorine components, a high heating equilibrium temperature is beneficial to reaching a distribution equilibrium state in the gas-solid phase as quickly as possible and increasing its concentration in the gas phase. However, temperatures above 100°C can easily lead to oxidation and Maillard caramelization of intrinsic substances in the Chinese medicinal material samples. To reduce the occurrence of these changes, the air in the headspace bottle is replaced with an inert gas such as isobaric nitrogen before and after sample packaging.

[0014] More preferably, a vacuum treatment is added after the deoxygenation treatment, and the vacuum treatment step is to connect a syringe to the headspace bottle after the cap is sealed, and extract gas in an amount greater than or equal to the volume of the headspace bottle.

[0015] The higher the temperature, the greater the gas pressure in the bottle. This requires a tighter seal, as leakage is more likely to occur. In the present invention, a vacuum is used to remove some of the gas in the sample bottle before constant-temperature heating of the headspace. This vacuum helps increase the concentration of the volatile organic fluorine component.

[0016] Preferably, the temperature of the headspace treatment is 100-150° C., and the equilibration time is 5-40 min.

[0017] Preferably, the solid phase microextraction needle of the carbon-containing molecular sieve material is a DVB / CAR / PDMS solid phase microextraction needle.

[0018] Preferably, the extraction treatment is carried out at a temperature of 40-90° C. and for a time of 30-70 min.

[0019] Preferably, the gas chromatograph of the gas chromatograph-mass spectrometer uses a non-polar, medium-polar or polar capillary chromatographic column for separation; the inlet temperature of the gas chromatograph is 250°C, the carrier gas is 99.999% helium, and the flow rate is 1.0 mL / min.

[0020] In the present invention, the chromatographic column can be a DB-5ms or TR-WAXMS chromatographic column with a specification of 30 m×0.25 mm×0.25 μm.

[0021] Preferably, the temperature program of the gas chromatography is: initial temperature is 40°C, maintained for 2 min, then heated to 240°C at 5°C / min, and maintained for 5 min.

[0022] Preferably, the mass spectrometry conditions of the gas chromatography-mass spectrometry instrument are: the ion source is an electron impact ionization source, the electron impact energy is 70 eV, the ion scanning range is 35-700 m / z, the ion source temperature is 240°C, and the transfer line temperature is 250°C.

[0023] Preferably, the organic fluorine is perfluorotributylamine, perfluorotripentylamine, polyfluoroalkane or polyfluoroalkyl ether.

[0024] Preferably, the method for establishing the standard curve of the organic fluorine is: the standard organic fluorine is prepared into a standard working solution of 2~75 mg / mL, detected by gas chromatography-mass spectrometry, and the standard curve is drawn with the concentration of the organic fluorine as the vertical coordinate and the corresponding peak area of ​​the organic fluorine as the horizontal coordinate.

[0025] Compared with the prior art, the present invention has the following beneficial effects: In this invention, high-temperature headspace heating combined with headspace treatment or conventional headspace heating combined with solid-phase microextraction (SPME) is used. By increasing the headspace heating temperature (100-200°C) of the sample, the volatilization separation rate and concentration of high-boiling-point, non-volatile organic fluorine components are increased. Alternatively, conventional headspace heating (below 100°C) combined with solid-phase microextraction (SPME) needle extraction and adsorption of organic fluorine using carbon-containing molecular sieve materials increases the separation and enrichment concentration of organic fluorine, thereby improving the sensitivity of gas chromatography-mass spectrometry for the determination of organic fluorine in traditional Chinese medicines. Combined with the calibration curve of organic fluorine standards, quantitative and qualitative analysis of organic fluorine in traditional Chinese medicines can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The total ion current chromatograms of the Cordyceps sinensis Chinese medicinal material samples of Example 1 and Comparative Example 1 are shown, wherein the upper figure is Comparative Example 1 and the lower figure is Example 1.

[0027] Figure 2 These are the total ion current chromatograms of three Cordyceps sinensis Chinese medicinal material samples with different origins and batch numbers in Example 2. The upper sample has an origin with batch number QRT20250129001, the middle sample has an origin with batch number PM20241029001, and the lower sample has an origin with batch number JZC20250328001.

[0028] Figure 3 These are the total ion current chromatograms of three Cordyceps sinensis Chinese medicinal material samples with different origins and batch numbers in Example 3, among which the origin and batch number in the upper figure is JX20240131001, the origin and batch number in the middle figure is QH20240131001, and the origin and batch number in the lower figure is LWF20240221001. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited to the following examples.

[0030] The raw materials used in the present invention are all commercially available.

[0031] To establish a standard curve, dilute the organic fluorine (perfluorotributylamine, perfluorotripentylamine) standards to prepare standard working solutions at concentrations of 2, 4, 6, 8, and 10 mg / mL. Place 100 μL of the solution in a 20 mL headspace vial and analyze using a gas chromatography-mass spectrometer under the following conditions: Carrier gas: helium with a purity of ≥99.999%; column flow rate: 1.0 mL / min; chromatographic column: TR-WAXMS (30 m × 0.25 mm × 0.25 μm); injection mode: split injection, split ratio 50:1; injection port temperature: 250°C; temperature program: 40°C for 2 min, then increase to 240°C at 5°C / min and hold for 5 min; mass spectrometer ion source temperature: 240°C; transfer line temperature: 250°C; electron impact energy: 70 eV; ion scan range (m / z): 35–700; The standard curve was drawn with the concentration of organic fluorine as the ordinate (Y) and the corresponding peak area of ​​organic fluorine as the abscissa (X). The results are shown in Table 1.

[0032] Table 1: Calibration curve equations and R values ​​for organic fluorine compounds 2

[0033] Example 1 (1) The sample material of Cordyceps sinensis (origin batch number: JZC20250129001) was crushed and sieved, and 200 mg of powder sample was taken into a 20 mL headspace bottle, sealed with a pressure cap, and a silicone polytetrafluoroethylene sealing gasket (resistant to high temperature of 200 ° C) was built into the hole in the middle of the aluminum cap. Two injection needles were inserted into the sealing gasket, one of which was connected to the atmosphere and the other was connected to the nitrogen cylinder pressure reducing valve or nitrogen blower. Nitrogen was passed through the bottle at normal pressure for 2 minutes to replace the air in the headspace bottle. After the replacement was completed, the needle connected to the nitrogen was first unplugged, and then the other needle connected to the atmosphere was immediately unplugged. The headspace bottle was incubated at a constant temperature of 140 ° C for 5 minutes; 1000 μL of gas sample was drawn using a headspace autosampler, and the temperature of the autosampler syringe was 145 ° C; (2) The gas collected in step (1) is analyzed by gas chromatography-mass spectrometry; The parameters of gas chromatography-mass spectrometry analysis are as follows: Carrier gas: helium with a purity of ≥99.999%; column flow rate: 1.0 mL / min; chromatographic column: TR-WAXMS (30 m × 0.25 mm × 0.25 μm); injection mode: split injection, split ratio 50:1; injection port temperature: 250°C; temperature program: 40°C for 2 min, then increase to 240°C at 5°C / min and hold for 5 min; mass spectrometer ion source temperature: 240°C; transfer line temperature: 250°C; electron impact energy: 70 eV; ion scan range (m / z): 35–700; Figure 1The figure below is a total ion current chromatogram of the Cordyceps sinensis Chinese medicinal material sample in Example 1. The chromatographic peaks of the Cordyceps sinensis Chinese medicinal material sample were searched through the mass spectrometry database NIST, and it was found that in addition to perfluorotributylamine and perfluorotripentylamine, it also contained a variety of other organic fluorine components such as polyfluoroalkyl ethers and polyfluoroalkanes. The mass spectrometry data can clearly identify them as organic fluorine. The molecular formula in Table 2 is the molecular formula of a similar structure obtained by searching the database.

[0034] Table 2: Mass spectrometry data of sample peaks in the total ion current chromatogram of the Cordyceps sinensis Chinese medicinal material sample of Example 1

[0035] The peak areas of all ion current peaks of organic fluorine components were combined and substituted into the standard curve of perfluorotributylamine or perfluorotripentylamine to calculate the organic fluorine content in the sample bottle. Divided by the mass of the Chinese medicinal material sample, the organic fluorine content of the sample was 5.8% (calculated as perfluorotributylamine).

[0036] Example 2 (1) The samples of Cordyceps sinensis from three different origins (origin batch numbers: QRT20250129001, PM20241029001, JZC20250328001) were crushed and sieved respectively, and 200 mg of powder sample was taken into 20 mL headspace bottles, which were sealed with a pressure seal. A silicone polytetrafluoroethylene gasket (resistant to high temperature of 200 °C) was built into the middle hole of the aluminum cap. Two injection needles were inserted into the gasket, one of which was connected to the atmosphere and the other was connected to the nitrogen cylinder pressure reducing valve or nitrogen blower. Nitrogen was passed through the headspace bottle at normal pressure for 2 minutes to replace the air in the headspace bottle. After the replacement was completed, the needle connected to the nitrogen was first unplugged, and then an airtight syringe was connected to the other needle to extract the gas in the 20 mL headspace bottle to generate negative pressure in the headspace bottle. The headspace bottle was then incubated at a constant temperature of 140 °C for 10 minutes. 1000 mL of nitrogen was drawn using a headspace autosampler. μL gas sample, the autosampler syringe temperature was 145 °C; (2) The gases collected in step (1) were analyzed by gas chromatography-mass spectrometry; the parameters of the gas chromatography-mass spectrometry analysis were the same as those in Example 1.

[0037] Figure 2 These are the total ion current chromatograms of three Cordyceps sinensis samples from different origins and batches in Example 2. The top image shows batch number QRT20250129001, the middle image shows batch number PM20241029001, and the bottom image shows batch number JZC20250328001. As shown, no organic fluorine components were detected in the first two batches, while the last batch contained a significant amount of 5.3% (calculated as perfluorotributylamine).

[0038] Example 3 (1) Three samples of Cordyceps sinensis from different origins (batch numbers: JX20240131001; QH20240131001; LWF20240221001) were crushed and sieved, and 200 mg of powder sample was taken from each sample into a 20 mL headspace bottle, which was sealed with a pressure seal. A DVB / CAR / PDMS solid phase microextraction needle (Supelco, 57348-U) was inserted into the headspace bottle through the sealing gasket, and the extraction needle was placed in the middle of the headspace bottle. The headspace bottle was incubated at 70 °C for 50 minutes, and the extraction needle was removed. (2) The samples in the extraction needle collected in step (1) were respectively analyzed by gas chromatography-mass spectrometry. The parameter conditions of gas chromatography-mass spectrometry analysis were as follows: Carrier gas: helium with a purity of ≥99.999%; column flow rate: 1.0 mL / min; chromatographic column: capillary gas chromatography column TR-WAXMS (30 m × 0.25 mm × 0.25 μm) with polyethylene glycol as stationary phase; inlet temperature: 250°C; temperature program: 40°C for 2 min, then increase to 240°C at a rate of 5°C / min and hold for 5 min; mass spectrometer ion source temperature: 240°C; transfer line temperature: 250°C; electron impact energy: 70 eV; ion scan range (m / z): 35–700.

[0039] Figure 3 These are the total ion current chromatograms of three Cordyceps sinensis samples from different origins in Example 3. The upper image is batch number JX20240131001, the middle image is batch number QH20240131001, and the lower image is batch number LWF20240221001. As shown, no organic fluorine components were detected in the samples with the middle and lower batch numbers, while the sample with the upper batch number contained a significant amount of organic fluorine, at 4.73% (calculated as perfluorotributylamine).

[0040] Comparative Example 1 The method of Comparative Example 1 is the same as that of Example 1, except that the temperature of the headspace treatment is 70°C.

[0041] Figure 1 The upper figure is the total ion current chromatogram of the Cordyceps sinensis Chinese medicinal material sample of Comparative Example 1. The measured organic fluorine content is mainly the low-boiling point and highly volatile organic fluorine components that appear earlier. The peak shapes of perfluorotributylamine and perfluorotripentylamine are very weak and the concentrations are low. As for the organic fluorine peaks that appear later, their peak shapes are even weaker and their concentrations are even lower.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for improving the sensitivity of GC-MS determination of organic fluorine in Chinese medicinal materials, characterized in that: The following steps are involved: (1) Sample pretreatment: After the Chinese medicinal materials are crushed, they are placed in a headspace bottle, sealed with a cap, and subjected to headspace treatment at 100-200 °C to collect the headspace gas; alternatively, a solid phase microextraction needle containing carbon molecular sieve material is used to perform extraction at a temperature below 100 °C and collect the sample in the extraction needle; (2) The headspace gas or the sample in the extraction needle collected in step (1) is detected using a gas chromatography-mass spectrometer, and the type and content of organic fluorine in the Chinese medicinal materials are analyzed based on the standard curve of organic fluorine established in advance.

2. The method for improving the sensitivity of GC-MS determination of organic fluorine in traditional Chinese medicine according to claim 1, characterized in that: Deoxygenation treatment is also performed before the headspace treatment.

3. The method for improving the sensitivity of determining organic fluorine in Chinese medicinal materials by GC-MS according to claim 2, characterized in that: The deoxygenation treatment step is to connect the nitrogen cylinder pressure reducing valve or nitrogen blowing device to the head space bottle after the pressure cap is sealed, and introduce nitrogen to replace the air in the head space bottle.

4. The method for improving the sensitivity of determining organic fluorine in Chinese medicinal materials by GC-MS according to claim 3, characterized in that: After the deoxygenation treatment, a vacuum treatment is added. The vacuum treatment step is to connect an injection syringe to the headspace bottle after the cap is sealed, and extract gas with an amount greater than or equal to the volume of the headspace bottle.

5. The method for improving the sensitivity of determining organic fluorine in Chinese medicinal materials by GC-MS according to claim 1, characterized in that: The temperature of the headspace treatment is 100-150°C, and the equilibration time is 5-40 minutes.

6. The method for improving the sensitivity of determining organic fluorine in Chinese medicinal materials by GC-MS according to claim 1, characterized in that: The solid phase microextraction needle of the carbon-containing molecular sieve material is a DVB / CAR / PDMS solid phase microextraction needle.

7. The method for improving the sensitivity of determining organic fluorine in Chinese medicinal materials by GC-MS according to claim 1, characterized in that: The extraction process is carried out at a temperature of 40-90°C and for a time of 30-70 minutes.

8. The method for improving the sensitivity of determining organic fluorine in Chinese medicinal materials by GC-MS according to claim 1, characterized in that: The gas chromatograph of the gas chromatograph-mass spectrometer uses a non-polar, medium-polar, or polar capillary column for separation; the gas chromatograph has an inlet temperature of 250°C, a carrier gas of 99.999% helium, and a flow rate of 1.0 mL / min; The temperature program of the gas chromatography was as follows: initial temperature was 40 °C, maintained for 2 min, then heated to 240 °C at 5 °C / min, maintained for 5 min; The mass spectrometry conditions of the gas chromatography-mass spectrometry instrument are as follows: the ion source is an electron impact ionization source, the electron impact energy is 70 eV, the ion scan range is 35-700 m / z, the ion source temperature is 240°C, and the transfer line temperature is 250°C.

9. The method for improving the sensitivity of determining organic fluorine in Chinese medicinal materials by GC-MS according to claim 1, characterized in that: The organic fluorine is perfluorotributylamine, perfluorotripentylamine, polyfluoroalkane or polyfluoroalkyl ether.

10. The method for improving the sensitivity of determining organic fluorine in traditional Chinese medicine by GC-MS according to claim 1, characterized in that: The method for establishing the standard curve of the organic fluorine is as follows: the standard organic fluorine is prepared into a standard working solution of 2-75 mg / mL, detected by gas chromatography-mass spectrometry, and the standard curve is drawn with the concentration of the organic fluorine as the vertical axis and the corresponding peak area of ​​the organic fluorine as the horizontal axis.

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