Method for vacuum-assisted headspace-solid phase microextraction of flavor substances in fish oil

Abstract

The invention discloses a method for vacuum-assisted headspace-solid phase microextraction of flavor substances in fish oil. The method comprises the following steps: preparing a vacuum headspace bottle; sample pretreatment; extracting fish oil flavor substances; and performing GC-MS (gas chromatography-mass spectrometry) analysis treatment. The method comprises the following steps: sampling, extracting and enriching, injecting a sample into a gas chromatograph-mass spectrometer through manual sample injection, effectively separating complex sample components through gas chromatography by virtue of gas chromatography-mass spectrometry detection to obtain a high-purity sample and a chromatogram of the flavor substances in the fish oil, and comparing the chromatogram with a mass spectrum library to obtain the flavor substances in the fish oil. Then the relative content of the flavor substances in the fish oil is calculated through a peak area normalization method, the purpose of relative quantitative analysis is achieved, and efficient and accurate extraction of the flavor substances in the fish oil is achieved. According to the method, the quantity of the detected volatile substances can be effectively increased, the response signal of the flavor substance is improved, and a means with accuracy and precision is provided for research on the flavor substance of the fish oil.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and particularly relates to a method for vacuum-assisted headspace-solid phase microextraction of fish oil flavor substances. Background Art

[0002] Headspace solid-phase extraction-gas chromatography (HS-SPME-GC) is widely used in food flavor analysis. In recent years, with the deepening of food quality research, HS-SPME-GC has attracted much attention due to its high sensitivity, solvent-free nature, and ease of operation.

[0003] Traditional extraction methods have numerous shortcomings in analyzing volatile components in fish oil. Some conventional solvent extraction methods are not only cumbersome and time-consuming, but also prone to introducing impurities that interfere with analytical results. Furthermore, their extraction efficiency is low, making it difficult to fully extract volatile compounds that are present in low concentrations in fish oil but contribute significantly to flavor. While HS-SPME-GC technology has been applied to fish oil analysis, existing research has primarily focused on atmospheric pressure conditions. During atmospheric pressure extraction, some volatile components are difficult to volatilize due to factors such as intermolecular forces and boiling point, resulting in a limited number of detected compounds and an inability to fully reflect the true flavor characteristics of fish oil. A vacuum environment can alter the system pressure, reducing the resistance to gas molecules escaping, theoretically allowing more volatile components to volatilize and be detected. However, limited research is currently focused on vacuum-assisted headspace solid-phase extraction (VASE) of volatile components in fish oil. This method faces challenges such as high equipment dependence, difficult maintenance, complex techniques, high equipment costs, significant interference from the sample matrix, and difficulty in method development and optimization. Consequently, the method remains immature. Therefore, developing an efficient vacuum-assisted headspace-solid phase microextraction method for fish oil flavor substances is of great significance for accurately analyzing the volatile components of fish oil and improving the research level of fish oil quality. Summary of the Invention

[0004] The present invention aims to address the shortcomings of conventional methods for extracting volatile substances from fish oil at atmospheric pressure, such as low efficiency, long time consumption, poor accuracy, and insufficient precision. A method for vacuum-assisted headspace-solid phase microextraction of flavor substances from fish oil is provided. This method can achieve qualitative and relative quantitative analysis of flavor substances in fish oil, as well as efficient and precise extraction. It can also effectively increase the amount of detected volatile substances and improve the flavor substance response signal, thereby providing a means with both accuracy and precision for the study of flavor substances in fish oil.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a method for vacuum-assisted headspace-solid phase microextraction of fish oil flavor substances, comprising the following steps:

[0006] (1) Preparation of vacuum headspace bottle

[0007] The headspace bottle is slightly covered with a fork plug and placed in a vacuum freeze dryer, and then the vacuum pump of the vacuum freeze dryer is turned on. After vacuuming, the vacuum pump is turned off, and the fork plug and the headspace bottle are pressed tightly to seal. Then, the headspace bottle is taken out, and a hollow aluminum cap is covered on the headspace bottle and pressed tightly for secondary sealing to prepare a vacuum headspace bottle;

[0008] (2) Sample pretreatment

[0009] Use a disposable syringe with a needle to draw an appropriate amount of fish oil and inject it into a vacuum headspace bottle;

[0010] (3) Extraction of fish oil flavor substances

[0011] The vacuum headspace bottle and the disposable syringe with a needle are placed in an incubator and equilibrated to the preset extraction temperature. The extraction needle is inserted through the fork plug on the vacuum headspace bottle into the injection port of the disposable syringe with a needle. Then, the activated SPME fiber head is used for headspace extraction to enrich the volatile substances in the fish oil. After the extraction is completed, the SPME fiber head is retracted and the extraction needle is pulled out to complete the extraction.

[0012] (4) GC-MS analysis and processing

[0013] The extraction needle is inserted into the inlet of a gas chromatography-mass spectrometer, and the SPME fiber head is extended for desorption. After desorption is completed, the SPME fiber head is retracted, the extraction needle is removed, and a chromatogram of the flavor substances in the fish oil is obtained by gas chromatography-mass spectrometry. The obtained chromatogram is compared with the mass spectrum library to confirm the chemical composition of the flavor substances in the fish oil, and then the relative content of the flavor substances in the fish oil is calculated by the peak area normalization method.

[0014] Preferably, in step (1), the fork plug is a 2-4 angle fork plug, and the vacuum pump is turned off after the vacuum is evacuated to 0-2 Pa.

[0015] Preferably, in step (2), the amount of fish oil absorbed is 1 to 3 mL.

[0016] Preferably, in step (3), the equilibration time is 20 to 40 minutes, the preset extraction temperature is 30 to 50° C., and the headspace extraction time of the activated SPME fiber is 15 to 25 minutes.

[0017] Preferably, in step (4), the chromatographic conditions of the gas chromatography-mass spectrometer are: DB-WAX capillary column, split ratio (20-30): 1; inlet temperature 250-260°C; detector temperature 250-260°C; carrier gas He, flow rate 1.0-1.5 mL / min; electron energy 60-70 eV; ion source temperature 230-240°C; quadrupole temperature 450-460°C; heating program: 50-55°C for 3-3.5 min, increased to 120-125°C at a rate of 8-9°C / min for 3-4 min, increased to 165-170°C at a rate of 4-5°C / min for 3-4 min, and increased to 220-240°C at a rate of 9-10°C / min for 2-3 min.

[0018] Compared with the existing technology, the present invention has the following advantages: the vacuum-assisted headspace-solid phase microextraction method of fish oil flavor substances of the present invention, after sampling, extraction, and enrichment, is manually injected into a gas chromatography-mass spectrometer. With the help of gas chromatography-mass spectrometry (GC-MS), the complex sample components are effectively separated by gas chromatography, obtaining a high-purity sample, and a chromatogram of the flavor substances in the fish oil is obtained. The obtained chromatogram is compared with a mass spectrum library to qualitatively identify the flavor substances in the fish oil. The relative content of the flavor substances in the fish oil is then calculated by peak area normalization, achieving the purpose of relative quantitative analysis and realizing efficient and accurate extraction of flavor substances in fish oil. The method of the present invention can effectively increase the number of volatile substances detected and improve the flavor substance response signal, providing a means for the study of fish oil flavor substances with both accuracy and precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a chromatogram of flavor substances in fish oil extracted in Example 1;

[0020] Figure 2 This is a chromatogram of flavor substances extracted from fish oil in Comparative Example 1;

[0021] Figure 3 The gas chromatography analysis results of the flavor substances in the fish oil extracted in Example 1 and Comparative Example 1 are shown. DETAILED DESCRIPTION

[0022] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0023] Taking fish oil produced by Ningbo Jinri Food Co., Ltd. as an example, the flavor substances contained in the fish oil were extracted using the methods of Example 1 and Comparative Example 1 below, and the extraction effects of volatile substances under vacuum and normal pressure were compared.

[0024] Example 1: A method for vacuum-assisted headspace-solid phase microextraction of fish oil flavor substances, comprising the following steps:

[0025] (1) Preparation of vacuum headspace bottle

[0026] A 12 mL headspace vial was slightly capped with a four-corner prong stopper and placed in a vacuum freeze dryer. The vacuum pump of the vacuum freeze dryer was then turned on and the vacuum was evacuated to 0-2 Pa. The vacuum pump was then turned off. The four-corner prong stopper and the headspace vial were pressed tightly to seal. The headspace vial was then removed and capped with a 20-thread hollow aluminum cap and pressed tightly with Agilent manual opening pliers for secondary sealing to prepare a vacuum headspace vial.

[0027] (2) Sample pretreatment

[0028] Use a disposable syringe with a needle to draw 2 mL of fish oil and inject it into the vacuum headspace bottle;

[0029] (3) Extraction of fish oil flavor substances

[0030] The vacuum headspace bottle and the disposable syringe with a needle were placed in an incubator and equilibrated for 30 minutes to a preset extraction temperature of 40°C. The extraction needle was inserted through the fork plug on the vacuum headspace bottle into the injection port of the disposable syringe with a needle. The activated SPME fiber head was then used for headspace extraction for 20 minutes to enrich the volatile substances in the fish oil. After the extraction was completed, the SPME fiber head was retracted and the extraction needle was pulled out to complete the extraction.

[0031] (4) GC-MS analysis and processing

[0032] The extraction needle was inserted into the inlet of a gas chromatography-mass spectrometer (model: Agilent 5877B GC / MSD), and the SPME fiber head was extended for desorption. After the desorption was completed, the SPME fiber head was retracted, the extraction needle was removed, and the chromatographic conditions of the gas chromatography-mass spectrometer were set as follows: DB-WAX capillary column, split ratio 20:1; inlet temperature 250°C; detector temperature 250°C; carrier gas He, flow rate 1.0 mL / min; electron energy 70 eV; ion source temperature 230°C; quadrupole temperature 450°C; heating program: 50°C for 3 min, increased to 125°C at a rate of 8°C / min and held for 3 min, increased to 165°C at a rate of 4°C / min and held for 3 min, increased to 230°C at a rate of 10°C / min and held for 2 min; the gas chromatography-mass spectrometer was used to obtain Figure 1 The chromatogram of the flavor substances in the fish oil is shown; the obtained chromatogram is compared with the mass spectrum library to confirm the chemical composition of the flavor substances in the fish oil, and then the relative content of the flavor substances in the fish oil is calculated by the peak area normalization method.

[0033] Comparative Example 1: A method for extracting fish oil flavor substances by headspace-solid phase microextraction at atmospheric pressure, comprising the following steps:

[0034] (1) Preparation of atmospheric pressure headspace bottle

[0035] Under indoor conditions, a 12 mL headspace vial was sealed with a four-corner prong stopper, and then a 20-thread hollow aluminum cap was placed on the vial and pressed tightly with an Agilent manual opener to perform a secondary seal, thereby producing a normal pressure headspace vial.

[0036] (2) Sample pretreatment

[0037] Use a disposable syringe with a needle to draw 2 mL of fish oil and inject it into the normal pressure headspace bottle;

[0038] (3) Extraction of fish oil flavor substances

[0039] The atmospheric pressure headspace bottle and the disposable syringe with a needle were placed in an incubator and equilibrated for 30 minutes to a preset extraction temperature of 40°C. The extraction needle was inserted through the fork plug on the atmospheric pressure headspace bottle into the injection port of the disposable syringe with a needle. The activated SPME fiber head was then used for headspace extraction for 20 minutes to enrich the volatile substances in the fish oil. After the extraction was completed, the SPME fiber head was retracted and the extraction needle was pulled out to complete the extraction.

[0040] (4) GC-MS analysis and processing

[0041] The extraction needle was inserted into the inlet of a gas chromatography-mass spectrometer (model: Agilent 5877B GC / MSD), and the SPME fiber head was extended for desorption. After the desorption was completed, the SPME fiber head was retracted, the extraction needle was removed, and the chromatographic conditions of the gas chromatography-mass spectrometer were set as follows: DB-WAX capillary column, split ratio 20:1; inlet temperature 250°C; detector temperature 250°C; carrier gas He, flow rate 1.0 mL / min; electron energy 70 eV; ion source temperature 230°C; quadrupole temperature 450°C; heating program: 50°C for 3 min, increased to 125°C at a rate of 8°C / min and held for 3 min, increased to 165°C at a rate of 4°C / min and held for 3 min, increased to 230°C at a rate of 10°C / min and held for 2 min; the gas chromatography-mass spectrometer was used to obtain Figure 2 The chromatogram of the flavor substances in the fish oil is shown; the obtained chromatogram is compared with the mass spectrum library to confirm the chemical composition of the flavor substances in the fish oil, and then the relative content of the flavor substances in the fish oil is calculated by the peak area normalization method.

[0042] Figure 3The gas chromatography analysis results of the flavor substances extracted in Example 1 under vacuum conditions and Comparative Example 1 under normal pressure are presented, namely, waterfall plots of the flavor substances in the fish oil extracted in Example 1 and Comparative Example 1 on the same coordinate axes. A comparison reveals that the retention times of the same components under vacuum and normal pressure are essentially the same, but there are differences in peak height and peak area, indicating that vacuum and normal pressure conditions affect the volatilization of components in the fish oil and the detection response. Retention time is primarily determined by the partition coefficient of a substance between the stationary phase and the mobile phase. When the chromatographic column is fixed and the temperature and other conditions are the same, the partition coefficient of a substance remains unchanged. Therefore, vacuum and normal pressure conditions do not significantly change the distribution behavior of the components in the fish oil within the chromatographic column, and the retention times are essentially the same. The reason for the difference in peak height and peak area is that the vacuum and normal pressure conditions of the headspace vial affect the volatilization of the components in the fish oil. Under a vacuum environment, the system pressure is reduced, making the substances more volatilizable, which can cause more fish oil flavor substances to volatilize, further affecting the detection signal. At the same time, there may be differences in the injection volume under vacuum and normal pressure conditions. The density of gas molecules in the headspace bottle under vacuum is low, and relatively more analytes may be introduced during injection, increasing the peak area and peak height; while the injection volume under normal pressure is relatively small, and the response signal is weak.

[0043] The specific component analysis results of the volatile substances in the fish oil extracted in Example 1 and Comparative Example 1 are shown in Table 1.

[0044] Table 1 Effects of vacuum and normal pressure on the composition and content of volatile substances

[0045]

[0046] As shown in Table 1, 20 and 15 volatile substances were detected in vacuum and normal pressure states, respectively, including 2 alcohols, 9 aldehydes, 7 alkanes, 1 heterocycle, and 1 ketone. There are differences in the types, relative contents, and peak areas of volatile substances under different extraction conditions. The volatility of substances under normal pressure and vacuum states is different. The vacuum state reduces the escape resistance of gas molecules by changing the system pressure, resulting in a 24.54±2.17-fold difference in the sum of the peak areas of the vacuum group and the normal pressure group. Taking syringaldehyde D, hexadecanal, and 4-ethylbenzaldehyde as examples, they were not detected in the normal pressure group (i.e., Comparative Example 1), but were detected in the vacuum group (i.e., Example 1), where the syringaldehyde D content was 2.57%±0.05%, and the content accounted for 23.63%±0.01% of the total volatile substances. This is because the vacuum environment overcomes the limitations of factors such as intermolecular forces and boiling point, creating volatilization conditions for these substances that are not easily volatile under normal pressure, thus enabling them to be detected. Substances were detected in both the normal pressure group and the vacuum group, but the content was different. For example, the content of myrtle alcohol in the normal pressure group was 1.50% ± 0.03%, and in the vacuum group it was 1.39% ± 0.30%. The principle is that pressure changes affect the gas-liquid equilibrium. For example, the gas-liquid equilibrium causes the proportion of substances such as (E,E)-2,4-heptadienal in the vacuum group to decrease slightly compared to its proportion in the normal pressure group. Under vacuum conditions, the system pressure decreases, and it is easier for substances to change from liquid to gas. The total amount of volatilization of the substance increases accordingly, which is ultimately reflected as a change in content in the test.

Claims

1. A method for vacuum-assisted headspace-solid phase microextraction of fish oil flavor substances, characterized in that: The following steps are involved: (1) Preparation of vacuum headspace bottle The headspace bottle is slightly covered with a fork plug and placed in a vacuum freeze dryer, and then the vacuum pump of the vacuum freeze dryer is turned on. After vacuuming, the vacuum pump is turned off, and the fork plug and the headspace bottle are pressed tightly to seal. Then, the headspace bottle is taken out, and a hollow aluminum cap is covered on the headspace bottle and pressed tightly for secondary sealing to prepare a vacuum headspace bottle; (2) Sample pretreatment Use a disposable syringe with a needle to draw an appropriate amount of fish oil and inject it into a vacuum headspace bottle; (3) Extraction of fish oil flavor substances The vacuum headspace bottle and the disposable syringe with a needle are placed in an incubator and equilibrated to the preset extraction temperature. The extraction needle is inserted through the fork plug on the vacuum headspace bottle into the injection port of the disposable syringe with a needle. Then, the activated SPME fiber head is used for headspace extraction to enrich the volatile substances in the fish oil. After the extraction is completed, the SPME fiber head is retracted and the extraction needle is pulled out to complete the extraction. (4) GC-MS analysis and processing The extraction needle is inserted into the inlet of a gas chromatography-mass spectrometer, and the SPME fiber head is extended for desorption. After desorption is completed, the SPME fiber head is retracted, the extraction needle is removed, and a chromatogram of the flavor substances in the fish oil is obtained by gas chromatography-mass spectrometry. The obtained chromatogram is compared with the mass spectrum library to confirm the chemical composition of the flavor substances in the fish oil, and then the relative content of the flavor substances in the fish oil is calculated by the peak area normalization method.

2. The method for vacuum-assisted headspace-solid phase microextraction of fish oil flavor substances according to claim 1, characterized in that: In step (1), the fork plug is a 2-4 angle fork plug, and the vacuum pump is turned off after vacuuming to 0-2 Pa.

3. The method for vacuum-assisted headspace-solid phase microextraction of fish oil flavor substances according to claim 1, characterized in that: In step (2), the amount of fish oil absorbed is 1 to 3 mL.

4. The method of vacuum-assisted headspace-solid phase microextraction of fish oil flavor substances according to claim 1, characterized in that: In step (3), the equilibration time is 20 to 40 minutes, the preset extraction temperature is 30 to 50° C., and the headspace extraction time of the activated SPME fiber is 15 to 25 minutes.

5. The method for vacuum-assisted headspace-solid phase microextraction of fish oil flavor substances according to claim 1, characterized in that: In step (4), the chromatographic conditions of the gas chromatography-mass spectrometer are: DB-WAX capillary column, split ratio (20-30): 1; inlet temperature 250-260°C; detector temperature 250-260°C; carrier gas He, flow rate 1.0-1.5 mL / min; electron energy 60-70 eV; ion source temperature 230-240°C; quadrupole temperature 450-460°C; heating program: 50-55°C for 3-3.5 min, increased to 120-125°C at a rate of 8-9°C / min for 3-4 min, increased to 165-170°C at a rate of 4-5°C / min for 3-4 min, and increased to 220-240°C at a rate of 9-10°C / min for 2-3 min.

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