Quantitative Detection Method for the Bitter Substance 7-Methoxycoumarin in Zanthoxylum Oil

Abstract

The present invention relates to the technical field of food analysis, and specifically to a quantitative detection method for 7-methoxycoumarin, a bitter substance in prickly ash oil, comprising the following steps: S1: adding a prickly ash oil sample to an organic solvent, followed by ultrasonic treatment and centrifugation in sequence, and subjecting the supernatant obtained after centrifugation to a first rotary evaporation to remove methanol to obtain a methanol extract; S2: dissolving the methanol extract in an ethanol solution, then extracting with petroleum ether, combining the petroleum ether phases after extraction, and subjecting to a second rotary evaporation to remove the organic solvent to obtain a petroleum ether extract; S3: redissolving and volume-fixing the petroleum ether extract with methanol, then passing through a microporous filter membrane, and the filtrate is the test solution; S4: detecting the test solution by high performance liquid chromatography. The detection method provided by the present invention has good accuracy, stability and repeatability, can be used for the quantitative analysis of 7-methoxycoumarin, provides a basis for the subsequent bitter taste regulation effect of prickly ash oil, and has important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of food analysis, and particularly to a quantitative detection method for the bitter substance 7-methoxycoumarin in prickly ash oil. Background Art

[0002] In addition to the fragrance and numbness, prickly ash produces an obvious bitterness during the consumption process. Whether it is the prickly ash itself or processed products such as prickly ash powder and prickly ash oil, they all have a certain degree of bitterness. The bitter substances in prickly ash are not the main components of prickly ash, but trace components and their synergistic effects. Among the components of the prickly ash pericarp, in addition to the main volatile aromatic oils and numbing substances, there are also substances such as flavonoids, alkaloids, and coumarins. Zhang Zhiqing et al. from the College of Food Science, Sichuan Agricultural University, first successfully identified coumarin as the main bitter component of prickly ash by using sensory-guided fractionation technology combined with UPLC-Q-TOF-MS method. Among them, 7-methoxycoumarin, as one of the coumarin categories, is the main bitter component of prickly ash. By activating the bitter receptor TAS2R14, it causes the release of neurotransmitters, and then produces bitterness in taste (Ke & Zhang et al., 2021). The research results were published in the international top food journal Food Chemistry and received extensive attention in this field. However, bitterness in food is usually not desired, and consumers will instinctively resist the bitterness in food, which largely leads to an unacceptable psychology of consumers towards bitter foods, and prickly ash and its processed products are no exception.

[0003] Prickly ash oil is one of the deep-processed products of prickly ash. It not only has the numbing fragrance characteristic of prickly ash, but also takes into account the nutritional value of vegetable oil. Moreover, it solves the problems of inconvenient cooking and eating of prickly ash grains and prickly ash powder, and effectively alleviates the adverse phenomena such as microbial contamination and loss of active ingredients during the storage and sales of prickly ash. However, for consumers, numbness and aroma are one of the key indicators for evaluating the edible quality of prickly ash oil. Therefore, in order to meet the market demand, manufacturers will improve the numbness and aroma of prickly ash oil by optimizing the production process of prickly ash oil, such as appropriately increasing the frying temperature and prolonging the frying time. Since the main bitter component of prickly ash is 7-methoxycoumarin, and it is a kind of fat-soluble compound, during the frying of prickly ash, this substance will gradually dissolve in the oil as the prickly ash cells rupture, thereby causing the prickly ash oil to produce an undesired bitterness. Bitterness will be reflected in the cooking and eating processes, and to a certain extent, it has an adverse impact on the taste and flavor of food. Therefore, bitterness has become a key quality problem of prickly ash oil.

[0004] In recent years, the bitterness of prickly ash oil has widely attracted the attention of the condiment industry, food processing industry, and catering industry. How to scientifically clarify the bitter substance 7-methoxycoumarin in prickly ash oil is the key to monitoring the product quality of prickly ash oil and the standard preparation of prickly ash oil industrialization. Based on this, exploring an effective detection method for the bitter substance 7-methoxycoumarin in prickly ash oil has become one of the urgent problems to be solved in this field. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a quantitative detection method for the bitter substance 7-methoxycoumarin in prickly ash oil, so as to achieve at least the effects of good stability, high precision, and good repeatability.

[0006] The purpose of the present invention is achieved by the following technical solutions:

[0007] A quantitative detection method for the bitter substance 7-methoxycoumarin in prickly ash oil, comprising the following steps:

[0008] S1: Add the prickly ash oil sample to an organic solvent, then perform ultrasonic treatment and centrifugation in sequence, and perform the first rotary evaporation on the supernatant obtained after centrifugation to remove methanol to obtain a methanol extract;

[0009] S2: Dissolve the methanol extract in an ethanol solution, then extract with petroleum ether, combine the petroleum ether phases after extraction, and perform the second rotary evaporation to remove the organic solvent to obtain a petroleum ether extract;

[0010] S3: Redissolve and make up the volume of the petroleum ether extract with methanol, then pass it through a microporous filter membrane, and the filtrate is the test solution;

[0011] S4: Detect the test solution by high performance liquid chromatography.

[0012] It should be understood that the prickly ash oil sample includes a flavoring oil with a numbing and fragrant smell obtained by directly soaking prickly ash in edible oil, or a prickly ash essential oil or prickly ash oleoresin prepared by methods such as organic solvent extraction and supercritical CO 2 extraction and a prickly ash blended oil with a special aroma and numbing feeling prepared by mixing with a certain proportion of edible oil. For example, for commercially available prickly ash oil products, as long as the bitter substance 7-methoxycoumarin in them is higher than the detection limit of the present invention, the method of the present invention can be used for detection.

[0013] Further, in step S1, the organic solvent includes at least one of methanol, ethanol, and acetonitrile;

[0014] And / or, 5-20 mL of the organic solvent is added to each gram of the prickly ash oil sample.

[0015] Further, in step S1, the temperature of the ultrasonic treatment is 25-40 °C;

[0016] And / or, the time of the ultrasound is 15 - 60 min.

[0017] Furthermore, in step S1, the centrifugation speed is 3000 - 5000 r / min;

[0018] And / or, the centrifugation time is 5 - 30 min.

[0019] Furthermore, in step S1, the temperature of the first rotary evaporation is 55 °C, the rotation speed is 120 r / min, and the rotary evaporation is carried out for 10 min.

[0020] Furthermore, in step S2, the mass fraction of the ethanol solution is 2.5% - 10%;

[0021] And / or, the ethanol solution is added at 6.25 - 25 mL per gram of the prickly ash oil sample.

[0022] Furthermore, in step S2, the petroleum ether is added at 18.75 - 75 mL per gram of the prickly ash oil sample.

[0023] Furthermore, in step S2, the extraction temperature is 25 - 35 °C.

[0024] Furthermore, in step S2, the temperature of the second rotary evaporation is 55 °C, the rotation speed is 120 r / min, and the rotary evaporation is carried out for 20 min.

[0025] Furthermore, in step S3, the pore size of the microporous filter membrane is 0.22 - 0.45 μm.

[0026] Furthermore, in step S4, the chromatographic conditions of the high performance liquid chromatography are as follows: an acetic acid solution with a volume concentration of 0.1% is used as mobile phase A, and acetonitrile with a volume concentration of 100% is used as mobile phase B, and mobile phase A and mobile phase B are mixed for gradient elution;

[0027] The gradient elution program is as follows: 0 - 5 min, mobile phase A decreases from 95% to 60%; 5 - 10 min, mobile phase A decreases from 60% to 50%; 10 - 15 min, mobile phase A decreases from 50% to 35%; 15 - 25 min, the proportion of mobile phase A remains at 35%; 25 - 30 min, mobile phase A increases from 35% to 95%.

[0028] It should be noted that the content of the bitter substance 7-methoxycoumarin in prickly ash oil is very low. In addition, there are a large number of phenolic substances in prickly ash oil. In the preliminary pre-experiment, it was found that the peak emergence times of a large number of substances were close, resulting in difficulty in separating substances. The present invention for the first time provides a high-performance liquid chromatography detection method for the bitter substance 7-methoxycoumarin in prickly ash oil, which can accurately detect the bitter substance 7-methoxycoumarin in prickly ash oil, calculate its content through the peak area of 7-methoxycoumarin, and monitor the product quality of prickly ash oil based on the content of 7-methoxycoumarin in prickly ash oil, and provide an intuitive judgment for the subsequent bitter taste regulation effect of prickly ash oil, having good application prospects.

[0029] The beneficial effects of the present invention are as follows:

[0030] The liquid chromatography detection method for the bitter substance 7-methoxycoumarin in prickly ash oil established by the present invention has a resolution greater than 1.3 for each peak under the chromatographic conditions of the present invention, can be effectively separated, and has good specificity; through precision determination, based on the results of 6 parallel operations, the RSD of the content of 7-methoxycoumarin in the sample is 0.17%, and the precision is good; through stability determination, within 0-24 h, the RSD of the content of 7-methoxycoumarin in the sample is 1.16%, and the stability is good; through repeatability determination, based on the results of 6 parallel operations, the RSD of the content of 7-methoxycoumarin in each sample is 4.75%, and the repeatability is good; under the conditions of low, medium, and high spiked concentrations, the recovery rate range of 7-methoxycoumarin is within 92.27% - 111.07%, and the relative deviation RSD is within 1.96% - 7.05%, indicating that the recovery rate results of the detection method of the present invention are good. The quantitative detection method for the bitter substance 7-methoxycoumarin in prickly ash oil established by the present invention can effectively and accurately detect the content of 7-methoxycoumarin, not only filling the research gap in the detection of the bitter substance 7-methoxycoumarin in prickly ash oil, but also based on the content of 7-methoxycoumarin in prickly ash oil, intuitively judging whether the bitter taste regulation method has a debittering effect, and having important guiding significance for the optimization of bitter taste regulation conditions. Description of the Drawings

[0031] Figure 1 It is the standard curve of 7-methoxycoumarin described in Example 1;

[0032] Figure 2 It is the HPLC chromatogram of the test solution described in Example 1;

[0033] Figure 3 It is the HPLC chromatogram of the test solution described in Comparative Example 1;

[0034] Figure 4 It is the HPLC chromatogram of the test solution described in Comparative Example 2;

[0035] Figure 5 It is the HPLC chromatogram of the spiked recovery test sample solution in Example 2;

[0036] Figure 6 It is the comparison chart of the determination results of five kinds of prickly ash oil samples in Example 3. Specific implementation manners

[0037] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the following.

[0038] The sources of some instruments and reagents used in the following examples are as follows:

[0039] Instruments: LC-2050C 3D high performance liquid chromatograph (Shimadzu Experimental Equipment Co., Ltd.); Sartorius CPA225D analytical balance (Sartorius AG, Germany); 2YMICRO-III-20T ultrapure water instrument (Sichuan Zhuoyue Water Treatment Equipment Co., Ltd.); KH-300DE ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instruments Co., Ltd.); HY-2 vortex mixer (Shanghai Yidian Scientific Instruments Co., Ltd.); Micromax centrifuge (Thermoflasher, USA); SHZ-D(III) circulating water multi-purpose vacuum pump (Gongyi Keri Instruments Co., Ltd.); RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instruments).

[0040] Reagents: Methanol (chromatographic grade), acetonitrile (chromatographic grade), and glacial acetic acid (chromatographic grade) are all purchased from Chengdu Kelong Chemical Reagent Factory; absolute ethanol (analytical grade) is purchased from Chengdu Jinshan Chemical Reagent Co., Ltd.; petroleum ether is purchased from Shanghai Titan Scientific Co., Ltd.; 7-methoxycoumarin standard (purity ≥ 98.00%) is purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0041] Example 1

[0042] 1) Chromatographic conditions: The chromatographic column is Shim-pack GIST C18 (4.6 mm × 250 mm, 5 μm), the detector is PDA, the mobile phase is 0.1% glacial acetic acid (A) - acetonitrile (B), the flow rate is 0.8 mL / min, the injection volume is 10 μL, the detection wavelength is 320 nm, and the column temperature is 40°C; the elution program settings are shown in Table 1 below:

[0043] Table 1 HPLC elution program

[0044] Time (min) 0.1% Glacial Acetic Acid (A%) Acetonitrile (B%) 0 95 5 5 60 40 10 50 50 15 35 65 25 35 65 30 95 5

[0045] 2) Preparation of the standard curve of 7-methoxycoumarin: Accurately pipette 0.001, 0.01, 0.025, 0.5, 2.5, and 5 mL of the reference stock solution at 0.1 mg / mL respectively, and make up the volume to 10 mL with chromatographic-grade methanol. Shake well to obtain a series of bitter component solutions with different concentration gradients. Perform the determination under the chromatographic conditions described in step (1), analyze the linear relationship between the peak area and the concentration of the standard solution, draw the standard curve, and fit to obtain the regression equation and correlation coefficient (r) corresponding to the standard compound. The results are shown in Figure 1 The regression equation of 7-methoxycoumarin is calculated as: y = 13051x + 6855.5 (R 2 = 0.9996). The test results of the standard curve are shown in Table 2:

[0046] Table 2 Test results of the standard curve

[0047] Standard Solution Concentration (μg / mL) Relative Retention Time (min) Peak Area 0.01 13.561 791 0.1 13.566 7281 0.25 13.541 18368 5 13.552 69111 25 13.554 336239 50 13.558 658082

[0048] 3) Preparation of the test sample solution: Weigh 4.0 g (accurate to 0.0001 g) of the prickly ash oil sample (purchased from Sichuan Wufeng Lihong Food Co., Ltd.) and place it in a 50 mL centrifuge tube. Add 40.0 mL of methanol, vortex for 1 min, then place it in an ultrasonic cleaner. The ultrasonic temperature is 30°C, and ultrasonic extraction is carried out for 30 min. Then centrifuge at a speed of 4500 r / min for 5 min. Take the supernatant and place it in a 250 mL stoppered conical flask, and remove the methanol solution by rotary evaporation (the temperature of the vacuum rotary evaporator is 55°C, the rotation speed is 120 r / min, and rotary evaporation is carried out for 10 min) to obtain the methanol extract. Then carry out extraction treatment: Add 50 mL of 2.5% ethanol solution to the flask and stir well to fully disperse the methanol extract. Extract with petroleum ether (keep the temperature in the range of 25 - 35°C during extraction), extract 3 times (3 × 50 mL), collect the petroleum ether phases and combine them in a 250 mL stoppered conical flask. Remove the organic solvent by a vacuum rotary evaporator (the temperature of the vacuum rotary evaporator is 55°C, the rotation speed is 120 r / min, and rotary evaporation is carried out for 20 min) to obtain the petroleum ether extract. Redissolve with methanol and make up the volume to 10 mL, then filter through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test sample solution.

[0049] 4) Perform the determination on the test sample solution under the chromatographic conditions described in step (1). The determination results are shown in Figure 2 .

[0050] Comparative Example 1

[0051] The difference between Comparative Example 1 and Example 1 is that extraction treatment is not carried out during the preparation process of the test sample solution.

[0052] (1) The chromatographic conditions are the same as those in Example 1;

[0053] (2) Preparation of the test solution: Weigh 4.0 g (accurate to 0.0001 g) of the prickly ash oil sample (purchased from Sichuan Wufeng Lihong Food Co., Ltd.) and place it in a 50 mL centrifuge tube. Add 40.0 mL of methanol, vortex for 1 min, then place it in an ultrasonic cleaner. The ultrasonic temperature is 30°C, and ultrasonic extraction is carried out for 30 min. Then centrifuge at a speed of 4500 r / min for 5 min. Take the supernatant and place it in a 250 mL stoppered conical flask. Rotavaporize (the temperature of the vacuum rotary evaporator is 55°C, the rotation speed is 120 r / min, and rotavaporize for 10 min) to remove the methanol solution to obtain the methanol extract. Re-dissolve it with methanol and make the volume up to 10 mL, and then filter through a 0.22 μm microporous filter membrane. Take the filtrate to obtain the test solution;

[0054] (3) Determine the test solution obtained in step (2) according to the chromatographic conditions described in step (1). The determination results are shown in Figure 3 .

[0055] Comparative Example 2

[0056] The difference between Comparative Example 2 and Example 1 is that mobile phase A and mobile phase B are isocratically eluted in a certain proportion.

[0057] (1) Chromatographic conditions: The chromatographic column is Shim-pack GIST C18 (4.6 mm × 250 mm, 5 μm), the detector is PDA, the mobile phase is 0.1% glacial acetic acid: acetonitrile (V / V = 35:65), the flow rate is 0.8 mL / min, the injection volume is 10 μL, the detection wavelength is 320 nm, and the column temperature is 40°C;

[0058] (2) The preparation of the test solution is the same as that in Example 1;

[0059] (3) Determine the test solution obtained in step (2) according to the chromatographic conditions described in step (1). The determination results are shown in Figure 4 .

[0060] It can be seen from the experimental results that in Comparative Column 1, since the extraction treatment step was not carried out during the preparation of the test solution, the separation of the detection results was poor, and the target peak could not be clearly identified. It was suspected that the impurity peak overlapped with the peak of 7-methoxycoumarin, affecting the detection effect. In Comparative Example 2, the elution time of 7-methoxycoumarin was earlier than that in Example 1, and the relative retention time was 4.878 min, but the resolution of the target peak was only 0.702. By establishing and optimizing the HPLC method for 7-methoxycoumarin, a bitter substance in prickly ash oil, the relative retention time of the target peak in Example 1 was 13.287 min. At the same time, through extraction treatment, the target component could be well separated from the impurities, and the resolution reached 1.362. In addition, this method could minimize the overlap of the peaks of other components with lower contents. Therefore, Example 1 was finally selected as the optimal choice of the present invention, which has reference significance for subsequent research.

[0061] Example 2

[0062] The HPLC methodology of the high performance liquid chromatography detection method in Example 1 was investigated. The precision, stability and repeatability of the method were evaluated by analyzing the relative standard deviation (RSD) of the relative peak area (RPA) and relative retention time (RRT).

[0063] (1) Precision experiment: Take the same sample solution, analyze it according to the chromatographic conditions in Example 1, inject samples continuously for 6 times, record the chromatogram, measure the peak area of 7-methoxycoumarin, and calculate the relative standard deviation (RSD) to investigate the precision of the instrument. The results are shown in Table 3.

[0064] (2) Stability experiment: Take the same sample solution, place it at room temperature, analyze it according to the chromatographic conditions in Example 1, inject samples at 0, 2, 4, 6, 8, 10, 12 and 24 h respectively, record the chromatogram, measure the peak area, and calculate the RSD to investigate the stability of the sample solution. The results are shown in Table 3.

[0065] (3) Reproducibility experiment: According to the sample solution preparation method, prepare 6 sample solutions in parallel, determine them according to the chromatographic conditions in Example 1, record the chromatogram, measure the peak area, and calculate the RSD to investigate the reproducibility of the method. The results are shown in Table 3.

[0066] Table 3 Investigation results of precision, stability and reproducibility of HPLC detection of 7-methoxycoumarin

[0067]

[0068] (4)Spiked recovery experiment: 7-Methoxycoumarin was added to the sample solution at three levels of low, medium, and high, with the addition amounts being 50%, 100%, and 150% of the original amount in the sample, respectively. Three spiked sample solutions were prepared and injected three times each. The peak areas of the target components were recorded, and the spiked recoveries and RSDs were calculated to investigate the accuracy of the method. The results are shown in Table 4 and Figure 5 .

[0069] Table 4 Results of the spiked recovery test for 7-methoxycoumarin

[0070]

[0071] The precision, stability, and repeatability of the HPLC detection method for the bitter substance 7-methoxycoumarin in prickly ash oil were investigated using the retention time, peak area of 7-methoxycoumarin, and the content calculated from the regression equation. The results are shown in Table 1. The precision of the method was detected by injecting a sample solution continuously six times. The results showed that the RSD (n = 6) of the precision of the retention time of the 7-methoxycoumarin chromatographic peak was 0.04%, the RSD (n = 6) of the peak area was 0.14%, and the RSD (n = 6) of the content was 0.17%, indicating good precision of the instrument. The same sample solution was placed at room temperature and detected under the same chromatographic conditions at 0, 2, 4, 8, 10, 12, and 24 h to evaluate the stability of the sample solution. The results showed that the RSD (n = 6) of the stability of the retention time of the 7-methoxycoumarin chromatographic peak was 0.10%, the RSD (n = 6) of the peak area was 0.93% - 0.95%, and the RSD (n = 6) of the content was 1.15% - 1.18%, indicating that the prickly ash oil sample was stable within 24 h after being prepared into a solution. The repeatability experiment of the method was to simultaneously prepare six sample solutions of the prickly ash oil sample in parallel and detect them under the same chromatographic conditions. The results showed that the RSD (n = 6) of the repeatability of the retention time of the 7-methoxycoumarin chromatographic peak was 0.02%, the RSD (n = 6) of the peak area was less than 3.78%, and the RSD (n = 6) of the content was less than 4.75%, indicating good repeatability of the method.

[0072] In the spiked experiment, 7-methoxycoumarin standard was added to the prickly ash oil sample at addition amounts of 50%, 100%, and 150% of the original amount in the sample to obtain three low, medium, and high spiked sample solutions. Each solution was injected three times, and the peak areas of the target components were recorded. The spiked recoveries and the corresponding RSD values were calculated. The results are shown in Table 2. The recoveries were in the range of 92.27% - 111.07%, and the RSD ≤ 7.05% (n = 3), indicating good spiked recoveries. As described above, the sample pretreatment method and the HPLC method of this experiment are effective and reliable.

[0073] Through methodological verification, the quantitative detection method of the bitter substance 7-methoxycoumarin in this prickly ash oil has the advantages of good accuracy, stability and repeatability, and can be used for quantitative analysis.

[0074] Example 3

[0075] Using the preparation method of the test solution and the chromatographic conditions in Example 1, the quantitative detection of the bitter substance 7-methoxycoumarin in several kinds of prickly ash oil on the market was carried out. The prickly ash oil of five brands, namely Wufeng Lihong, Chuanzhen, Shushangxian, Yongfenghe, and Yaomazi, were tested respectively. The experimental results are shown in Table 5 and Figure 6 .

[0076] Table 5 Test results of the standard curve

[0077] Number Brand 7-Methoxycoumarin Content (μg / g) 1 Wufeng Lihong 9.2473 2 Shangshuxian 4.7768 3 Chuanzhen 13.3815 4 Yongfenghe 14.8247 5 Yaomazi 8.5386

[0078] By using the HPLC quantitative method to determine the content of 7-methoxycoumarin in prickly ash oil of different brands, it can be found that the difference in the content of 7-methoxycoumarin is very large. From the data, it can be concluded that the prickly ash oil from Yongfenghe has the highest content of 7-methoxycoumarin, up to 14.8247 μg / g. Followed by the prickly ash oil from Chuanzhen, with a content of up to 13.3815 μg / g. While the content of 7-methoxycoumarin in the prickly ash oil from Shushangxian is the lowest, which is 4.7768 μg / g.

[0079] The above are only the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be changed within the scope of the concept described herein through the above teachings or the techniques or knowledge in related fields. And the changes and modifications made by those skilled in the art without departing from the spirit and scope of the present invention should all be within the protection scope of the appended claims of the present invention.

Claims

1. A quantitative detection method for the bitter substance 7-methoxycoumarin in pepper oil, characterized in that: The following steps are involved: S1: adding methanol to the pepper oil sample, then performing ultrasound and centrifugation in sequence, and performing a first rotary evaporation on the supernatant obtained after centrifugation to remove methanol to obtain a methanol extract; S2: dissolving the methanol extract in an ethanol solution, then extracting with petroleum ether, combining the extracted petroleum ether phases, and removing the petroleum ether through a second rotary evaporation to obtain a petroleum ether extract; S3: The petroleum ether extract is redissolved with methanol and fixed to volume, and then filtered through a microporous filter membrane, and the filtrate is the test solution; S4: Detecting the test solution by high performance liquid chromatography; And / or, adding 5-20 mL of methanol per gram of the pepper oil sample; In step S4, the chromatographic conditions of the high performance liquid chromatography are: using a C18 column, using a glacial acetic acid solution with a volume concentration of 0.1% as mobile phase A, and acetonitrile with a volume concentration of 100% as mobile phase B, and the mobile phase A and the mobile phase B are mixed for gradient elution; The gradient elution procedure is as follows: 0-5 min, mobile phase A is reduced from 95% to 60%; 5-10 min, mobile phase A is reduced from 60% to 50%; 10-15 min, mobile phase A is reduced from 50% to 35%; 15-25 min, the proportion of mobile phase A is maintained at 35%; 25-30 min, mobile phase A is increased from 35% to 95%; The detection wavelength of the high performance liquid chromatography is 320 nm.

2. The detection method according to claim 1, characterized in that: In step S1, the temperature of the ultrasound is 25-40°C; And / or, the ultrasonic time is 15-60 min.

3. The detection method according to claim 1, characterized in that: In step S1, the centrifugal speed is 3000-5000 r / min; And / or, the centrifugation time is 5-30 min.

4. The detection method according to claim 1, characterized in that: In step S2, the mass fraction of the ethanol solution is 2.5%-10%; And / or, 6.25-25 mL of the ethanol solution is added per gram of the pepper oil sample.

5. The detection method according to claim 1, characterized in that: In step S2, 18.75-75 mL of petroleum ether is added per gram of the pepper oil sample.

6. The detection method according to claim 1, characterized in that: In step S2, the extraction temperature is 25-35°C.

7. The detection method according to claim 1, characterized in that: In step S3, the pore size of the microporous filter membrane is 0.22-0.45 μm.

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