Method for simultaneously detecting six chemical substances in natural perfume

Abstract

The invention belongs to the technical field of analysis and detection of natural perfume, and particularly relates to a method for simultaneously detecting six chemical substances in natural perfume. The detection method comprises the following steps: (1) carrying out mixed extraction on natural perfume by using an internal standard extraction agent to prepare a sample solution to be detected; (2) carrying out gas chromatography-mass spectrometry analysis on the sample solution to be detected, and respectively obtaining the contents of ethyl acrylate, pyridine, myrcene, longifolenone, benzophenone and methyleugenol according to the standard curve and the mass spectrum; a chromatographic column for the gas chromatography-mass spectrometry analysis is a weak-polarity stationary phase chromatographic column. According to the method disclosed by the invention, the to-be-detected target object can be efficiently extracted by selecting the styrax propionate and the dichloromethane as internal standard extraction agents, accurate analysis of the six chemical substances is realized by combining a weak-polarity stationary phase chromatographic column, matrix interference is eliminated, and the detection accuracy is high. And simultaneous determination of the six chemical substances in different dosage forms such as tincture, extract, essential oil, absolute oil and the like and the natural perfume with matrix components is realized.

Description

Technical Field

[0001] This invention belongs to the field of analytical testing technology of natural fragrances, specifically relating to a method for simultaneous detection of six chemical substances in natural fragrances. Background Technology

[0002] As people's living standards continue to improve, the safety risks of natural flavorings and their impact on human health have attracted increasing attention from researchers. Due to increasingly stringent food safety requirements, many functional chemical components frequently used in the food industry have been found to pose potential risks such as toxicity and carcinogenicity after more in-depth toxicological studies. In 2018, the U.S. Food and Drug Administration (FDA) banned the separate addition of chemicals such as ethyl acrylate, myrcene, pyridine, pulegone, methyl eugenol, and benzophenone.

[0003] Although the current national food safety standard GB 2760-2024, which is in effect in my country, does not completely prohibit the addition of these six compounds, sufficient preparations should be made in advance for the development of detection methods for these chemical substances, especially for chemical substances that pose safety risks.

[0004] Currently, there are some literature reports on the detection methods for these six compounds, but most of them focus on one of the compounds and its homologues. For example, Li Guanlin et al. studied the simultaneous determination of methyl acrylate, ethyl acrylate and propyl acrylate in air by gas chromatography (Energy and Environment, 2018, 4, 32-33); Wang Hongsong et al. studied the simultaneous determination of benzophenone and 4-methylbenzophenone in simulated sunflower seed oil liquid in food packaging by gas chromatography-mass spectrometry (Analytical Laboratory, 2011, 30(5): 39-41); Gao Ping et al. used SPE-GC / MS external standard method to determine four eugenol anesthetics in aquatic products (Food Industry Technology, 2019, 40(18): 229-233).

[0005] Some detection methods are only applicable to samples of the same type, making it difficult to meet the detection requirements of complex categories of natural fragrances. For example, Wang Xiulan et al. studied the determination of myrcene, 2-nonanone, isoborneol, and turmeric alcohol in turmeric volatile oil by gas chromatography (Journal of Shandong University of Traditional Chinese Medicine, 2019, 43(5): 513-518); Li Yan et al. used GC to determine the content of menthol in vanilla volatile oil (Journal of Xinjiang Medical University, 2017, 40(09): 1136-1137+1141); the natural fragrances analyzed by these existing technologies are all volatile oils (essential oils), and the interference from non-volatile matrices is minimal. In addition, Fang Yu et al. studied the determination of pyridine in water by purge-and-trap gas chromatography (Chemical and Biological Engineering, 2025, 42(02): 63-65+68).

[0006] Due to the different properties of ethyl acrylate, pyridine, myrcene, menthone, benzophenone, and methyl eugenol, it is difficult to detect these six chemical substances using the same method. Furthermore, natural fragrances come in various types, such as tinctures, extracts, essential oils, and absolutes, and the interference from different matrices varies considerably. Currently, there is no universal method to simultaneously detect and analyze ethyl acrylate, pyridine, myrcene, menthone, benzophenone, and methyl eugenol in different types of natural fragrances, which hinders efficient detection and risk management of these key substances. Summary of the Invention

[0007] The purpose of this invention is to provide a method for the simultaneous detection of six chemical substances in natural fragrances, in order to solve the problem that existing methods cannot simultaneously determine ethyl acrylate, pyridine, myrcene, menthone, benzophenone and methyl eugenol in different types of natural fragrances.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for simultaneous detection of six chemical substances in a natural fragrance, comprising the following steps: (1) The natural fragrance was mixed and extracted using an internal standard extractant to prepare the sample solution to be tested; the internal standard extractant was composed of styrax propionate and dichloromethane; (2) The sample solution to be tested is subjected to gas chromatography-mass spectrometry analysis. Based on the standard curve and mass spectrum, the contents of ethyl acrylate, pyridine, myrcene, longleaf menthone, benzophenone and methyl eugenol are obtained respectively. The chromatographic column of the gas chromatography-mass spectrometry analysis is a weakly polar stationary phase chromatographic column.

[0009] This invention is pioneering. By selecting styrax propionate and dichloromethane as internal standard extractants, the target analytes can be extracted efficiently. Combined with a weakly polar stationary phase chromatographic column, the above six chemical substances can be accurately analyzed, eliminating matrix interference and enabling the simultaneous determination of the above six chemical substances in natural fragrances of different dosage forms and matrix components, such as tinctures, extracts, essential oils, and absolutes.

[0010] This invention eliminates the need for different pretreatment and detection methods for different samples, enabling the simultaneous separation and detection of ethyl acrylate, pyridine, myrcene, menthone, benzophenone, and methyl eugenol. This avoids the cumbersome steps of detecting each of the six components separately, improves detection efficiency, saves consumables and reagents, and is more environmentally friendly.

[0011] Under optimized detection parameters, this method achieves high accuracy and precision. The linear exponents of the regression equations are all greater than 0.99, the relative standard deviation (RSD) is between 0.49% and 4.17%, and the average recovery rate is between 80.96% and 119.79%, which proves the detection effectiveness of this method.

[0012] Preferably, the weakly polar stationary phase column is a DB-5ms.

[0013] Preferably, the internal standard extractant consists of 10-50 mg of styrax ethyl propionate and 1-5 L of dichloromethane. More preferably, the internal standard extractant consists of 20-30 mg of styrax ethyl propionate and 2-3 L of dichloromethane. The above proportions can be increased or decreased proportionally.

[0014] More preferably, the amount of the natural fragrance is 10-200 mg, corresponding to a dosage of 25-50 mL of the internal standard extractant. Most preferably, the amount of the natural fragrance is 100-110 mg, corresponding to a dosage of 25-30 mL of the internal standard extractant.

[0015] Preferably, the mixed extraction further includes drying with anhydrous sodium sulfate. More preferably, the mass ratio of anhydrous sodium sulfate to natural fragrance is 1:1 to 9.

[0016] Preferably, the natural fragrance is a tincture, extract, essential oil, or absolute oil.

[0017] Preferably, the gas chromatography-mass spectrometry analysis conditions are as follows: carrier gas: helium; column flow rate: 1 mL / min; injection port temperature: 250℃; temperature program: 50℃, 0 min, 5℃ / min → 250℃, 50 min, hold at 250℃ for 10 min, then run at 260℃ for 10 min; split mode: splitless; transfer line temperature: 250℃; EI ion source temperature: 230℃, quadrupole temperature: 150℃; EI ionization energy: 70 eV; selected ion mode.

[0018] The detection method of this invention can be applied to the component detection of different types of natural fragrances or mixed fragrances of multiple natural fragrances. It is a general method for simultaneously detecting ethyl acrylate, pyridine, myrcene, menthone, benzophenone and methyl eugenol. It can achieve simultaneous quantitative detection of six substances, thereby providing technical support and data support for the safety risk management of natural fragrances. Attached Figure Description

[0019] Figure 1 The TIC charts are for the six compound standards in Example 1 of this invention. Figure 2 The TIC chromatograms are shown for the detection of six compound standards using different chromatographic columns in Comparative Example 1 of this invention. Figure 3 The TIC graphs are shown for the detection of six compound standards under different solvent conditions in Comparative Example 2 of this invention. Detailed Implementation

[0020] (a) Preferred embodiment of the method for simultaneous detection of six chemical substances in natural fragrances of the present invention Natural fragrance matrices are complex and diverse, and the structures of the six chemical substances mentioned above vary considerably. Existing testing methods are difficult to simultaneously cover the detection of these six chemical components. Therefore, this invention establishes a method for the simultaneous detection of ethyl acrylate, pyridine, myrcene, longleaf menthone, benzophenone, and methyl eugenol, which has significant practical implications.

[0021] This method, through pretreatment of natural fragrances and optimization of chromatographic columns for gas chromatography-mass spectrometry analysis, enables the simultaneous detection of ethyl acrylate, pyridine, myrcene, longleaf menthone, benzophenone, and methyl eugenol in samples. It overcomes the interference of complex matrices in natural fragrances and avoids the cumbersome experimental steps required by current methods to detect six substances separately, thus improving detection efficiency.

[0022] Using styrax propionate and dichloromethane as the internal standard extractant can improve baseline stability, avoid spurious peaks caused by column bleed, and facilitate accurate quantification. Using a weakly polar stationary phase column, such as the DB-5ms column, avoids problems such as uneven baselines, strong background signals, and excessive matrix interference that occur with other types of columns. It also ensures that the retention times of all six compounds are within 25 minutes, which helps to shorten instrument analysis time and improve instrument efficiency in sample analysis.

[0023] The preferred embodiment of the method for simultaneous detection of six chemical substances in the above-mentioned natural fragrances is as follows: (1) Accurately weigh a certain amount of styrax propionate, dilute to volume with dichloromethane as internal standard stock solution, and mix well for later use.

[0024] The internal standard stock solution can be used as an internal standard extractant for the direct extraction of natural fragrances. The mass concentration of styrax ester propionate in the internal standard stock solution is 1–50 mg / L.

[0025] Weigh out 10-50 mg of styrax propionate and dilute to 1-5 L with dichloromethane.

[0026] (2) Accurately weigh the natural fragrance sample into a 50 mL conical flask, add 25 mL of internal standard extractant, add 0.1~0.5 g of anhydrous sodium sulfate to dry, place on a shaker for 30 min to mix, let stand for 10 min, take the supernatant and filter 1 mL into a brown chromatographic bottle to obtain the sample solution to be tested.

[0027] This step involves extracting and preparing the sample solution for testing. The amount of natural fragrance sample weighed can be 10–200 mg. The mass ratio of anhydrous sodium sulfate to the natural fragrance sample is 1:1–9. A 0.45 μm filter membrane can be used for filtration.

[0028] (3) Perform gas chromatography-mass spectrometry analysis on the sample solution to be tested. Based on the predetermined standard curve and the obtained mass spectrum of the sample to be tested, the contents of ethyl acrylate, pyridine, myrcene, longleaf menthone, benzophenone and methyl eugenol are obtained respectively.

[0029] The instrument conditions for gas chromatography-mass spectrometry (GC-MS) analysis included: DB-5ms column; carrier gas: helium; column flow rate: 1 mL / min; injection port temperature: 250℃; temperature program: 50℃ (0 min), 5℃ / min → 250℃ (50 min), hold at 250℃ for 10 min, then run at 260℃ (10 min). Split mode: splitless; transfer line temperature: 250℃; EI ion source temperature: 230℃, quadrupole temperature: 150℃; EI ionization energy: 70 eV.

[0030] During the above temperature ramping process, the initial temperature is 50℃, then the temperature is increased to 250℃ at a ramping rate of 5℃ / min (taking 50 min), and held at 250℃ for 10 min. The run after 260℃ does not involve a temperature ramping program; this is a routine procedure for cleaning the column and injection port.

[0031] The DB-5ms column parameters were 60 m × 0.25 mm × 0.25 μm. The standard curve was detected using selected ion mode (SIM). The SIM method parameters are shown in Table 1.

[0032] Table 1. Retention times and selected ions of six analyte compounds and internal standard.

[0033] In the SIM mode analysis above, the target compound is identified by two ions, qualitative ion 1 and qualitative ion 2. In the TIC plot of the SIM mode, if the detection of two ion peaks is satisfied at the corresponding retention time, then it is confirmed to be the target compound.

[0034] The instruments and reagents used in this invention are all available in laboratories with testing capabilities. The method is simple and easy to operate, and has good detection accuracy and high precision, making it of great practical significance.

[0035] The preferred embodiments are illustrated below with reference to specific examples. Unless otherwise specified, the raw materials involved in the embodiments are all conventional commercial raw materials, and the processing methods involved are all conventional methods in the art.

[0036] Example 1 The method for simultaneous detection of six chemical substances in natural fragrances in this embodiment adopts the following steps: S1. Preparation of a series of standard working solutions of six compounds Accurately weigh 20 mg of styrax propionate and dilute to 2 L with dichloromethane as an internal standard stock solution. Mix well and set aside.

[0037] Accurately weigh 25.0 mg each of ethyl acrylate, pyridine, myrcene, longleaf menthone, benzophenone, and methyl eugenol. Dilute to 250 mL with dichloromethane containing the internal standard, shake well, and obtain a standard stock solution of 0.10 mg / mL. Store in a sealed container at 4°C protected from light. The shelf life is 6 months.

[0038] Pipette 0.1, 0.2, 0.5, 1, 2, 5, and 10 mL of the standard stock solution respectively, and dilute to 10 mL with dichloromethane (internal standard stock solution) containing the internal standard. Shake well to obtain standard stock solutions of gradient concentrations.

[0039] S2. Standard curve regression equation, limit of detection, and limit of quantitation The standard stock solutions with the above gradient concentrations were analyzed by GC-MS in ascending order of concentration.

[0040] The instrument conditions for gas chromatography-mass spectrometry (GC-MS) analysis were as follows: Column: DB-5ms column; Carrier gas: Helium; Column flow rate: 1 mL / min; Injector temperature: 250℃; Temperature program: 50℃ (0 min), 5℃ / min → 250℃ (50 min), hold at 250℃ for 10 min, then run at 260℃ (10 min). Split mode: Splitless; Transfer line temperature: 250℃; EI ion source temperature: 230℃; Quadrupole temperature: 150℃; EI ionization energy: 70 eV.

[0041] TIC (Total Ion Chromatogram) of a series of standard working solutions of corresponding concentrations were obtained. Based on the peak areas of the target compound and its corresponding internal standard compound, the peak area ratio of the target compound peak to its corresponding internal standard peak was linearly fitted with the concentration to obtain a linear regression equation. The lowest concentration standard solution in the concentration range was injected 10 times consecutively, and the standard deviation (SD) of the peak area was calculated. The limit of detection and the limit of quantitation were obtained by calculating 3 times (3SD) and 10 times (10SD) of the obtained standard deviation based on the standard curve, as shown in Table 2.

[0042] Table 2. Linear regression equations, detection limits, and quantitation limits for the analytical methods.

[0043] As shown in Table 2, the linear exponents of the regression equations are all greater than 0.99, and the relative standard deviation (RSD) is between 0.49% and 4.17%, indicating that the method provided by this invention has high accuracy and precision.

[0044] S3. Model Sample Solution Testing and Analysis Accurately weigh 25 mg each of ethyl acrylate, pyridine, myrcene, longleaf menthone, benzophenone and methyl eugenol standard substances, dilute to 1 L with dichloromethane, shake well, and use as the model sample solution.

[0045] Weigh 100 mg of the above model sample solution into a 50 mL Erlenmeyer flask, add 25 mL of internal standard extractant, add 0.1 g of anhydrous sodium sulfate to dry, place on a shaker for 30 min to mix, let stand for 10 min, take the supernatant, filter 1 mL through a 0.45 μm filter membrane into a brown chromatographic bottle, and use it as the sample solution to be tested.

[0046] The sample solution to be tested was analyzed by gas chromatography-mass spectrometry, with the instrument parameters and methods the same as in step S2. The analysis was based on the standard curve and the obtained TIC chromatogram of the sample. Figure 1This allows for convenient calculation of the contents of ethyl acrylate, pyridine, myrcene, menthone, benzophenone, and methyl eugenol.

[0047] Example 2 The method for simultaneous detection of six chemical substances in natural fragrances in this embodiment is basically the same as that in Example 1, with the only difference being: S3. Testing of actual natural flavorings.

[0048] The selected natural fragrance samples covered different categories and physical states, including essential oils, extracts, absolutes, and tinctures. The test sample solutions were prepared in the same manner as in step S3 of Example 1: 100 mg of each type of natural fragrance sample was weighed into a 50 mL Erlenmeyer flask, 25 mL of internal standard extractant was added, followed by 0.1 g of anhydrous sodium sulfate (dried), and the mixture was shaken for 30 min to mix. After standing for 10 min, the supernatant was collected and filtered through a 0.45 μm filter membrane to obtain 1 mL of the test sample solution in a brown chromatographic bottle.

[0049] The sample solutions were subjected to GC-MS detection in the same manner as in Example 1 to obtain sample chromatograms. The peak areas of the six compounds and the internal standard in the sample were obtained from the sample chromatograms. Based on the peak areas and the regression equation of the standard curve obtained in Example 1, the contents of ethyl acrylate, pyridine, myrcene, longleaf menthone, benzophenone and methyl eugenol in the sample solution were calculated using the internal standard method. The detection results are shown in Table 3, where Nd represents not detected.

[0050] Table 3. Test results of six components in different types of natural fragrance samples.

[0051] Example 3 This example describes the determination of spiked recovery rates for four different dosage forms of natural flavorings.

[0052] The selected samples were laurel leaf essential oil, tree moss extract, oakmoss essential oil, and Angelica pubescens tincture.

[0053] Accurately weigh 100 mg of sample into a 50 ml Erlenmeyer flask, add 10 mL of the internal standard stock solution from Example 1, and add 50 μL, 100 μL and 200 μL of standard stock solutions of three concentrations respectively. Then add 0.1 g of anhydrous sodium sulfate, mix on a shaker for 30 min, let stand for 10 min, and then filter 1 ml of the supernatant into a brown chromatographic bottle as the test solution.

[0054] The test solution was subjected to GC-MS detection using the same parameters and methods as in Example 1 to obtain the sample chromatogram. The peak areas of the six compounds and the internal standard in the test solution were obtained from the chromatogram. Based on the peak area and the regression equation of the standard curve obtained in Example 1, the contents of ethyl acrylate, pyridine, myrcene, longleaf menthone, benzophenone and methyl eugenol in the test sample solution were calculated using the internal standard method. The spiked recovery rate was calculated based on the obtained contents of each substance. Table 4 shows the spiked recovery rate results.

[0055] Table 4. Spike recovery results for different samples

[0056] As shown in Table 4, the average recovery rate of this method for different types of samples such as essential oils, extracts, absolutes, and tinctures is between 80.96% and 119.79%, indicating that the method provided by this invention has high accuracy and precision.

[0057] (ii) Comparative Example Comparative Example 1 This comparative example shows the comparison of detection results using different chromatographic columns under the same experimental conditions.

[0058] The sample to be tested is the sample solution from Example 1.

[0059] The comparative examples used DB-WAX and INNO-WAX columns, which are commonly used for detecting volatile components in fragrances, with the preferred DB-5ms column serving as a control.

[0060] The test sample solution was analyzed by GC-MS using the same parameters and methods as in Example 1, and the TIC chromatogram of the test sample solution was obtained. Figure 2 ).

[0061] Depend on Figure 2 It is evident that the INNO-WAX column suffers from issues such as uneven baseline, strong background signal, and significant matrix interference. Compared to the DB-WAX column, it also failed to improve analytical performance. The DB-5ms column effectively addresses these problems. Furthermore, when using the DB-5ms column, the retention times of all six compounds were within 25 minutes, thus shortening instrument analysis time and improving instrument efficiency in sample analysis.

[0062] Comparative Example 2 This comparative example shows a comparison of detection results for different solvents under the same experimental conditions.

[0063] The main difference in the comparative example is that all operations using dichloromethane in Example 1 were replaced with acetone or methanol, respectively. The preparation methods, pretreatment methods, GC-MS detection parameters and methods for all other working solutions and test sample solutions were completely consistent with those described in Example 1, resulting in the acquisition of TIC chromatograms for the test sample solutions. Figure 3 ).

[0064] Depend on Figure 3 It can be seen that the chromatographic resolution is good under the three different solvent conditions, indicating that the DB-5ms column is the preferred analytical method. However, when using acetone and methanol as solvents, the baseline stability is relatively poor, and a small number of impurity peaks due to column bleed appear. Therefore, using dichloromethane as solvent is the preferred detection method.

[0065] As can be seen from the above, the detection method of the present invention has good accuracy and high precision, and is less affected by background signals and impurities. It can be used for the simultaneous detection of ethyl acrylate, pyridine, myrcene, longleaf menthone, benzophenone and methyl eugenol, thereby creating favorable conditions for the efficient detection and safe control of the above-mentioned key chemical substances.

[0066] Finally, it should be noted that the above description is only 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneous detection of six chemicals in a natural flavor, characterized by, The method comprises the following steps: (1) mixing and extracting the natural flavor with an internal standard extractant to prepare a sample solution; the internal standard extractant is composed of benzoin propionate and dichloromethane; (2) performing gas chromatography-mass spectrometry analysis on the sample solution, and obtaining the content of ethyl acrylate, pyridine, myrcene, pulegone, benzophenone and methyl eugenol according to a standard curve and a mass spectrum; the chromatographic column used in the gas chromatography-mass spectrometry analysis is a weak polar stationary phase chromatographic column.

2. The method for simultaneous detection of six chemicals in natural flavors according to claim 1, wherein, The weak polar stationary phase chromatographic column is a DB-5ms.

3. The method for simultaneous detection of six chemicals in natural flavors according to claim 1, wherein, The internal standard extractant is composed of 10-50 mg of benzoin propionate and 1-5 L of dichloromethane.

4. The method for simultaneous detection of six chemicals in natural flavors according to claim 3, wherein The internal standard extractant is composed of 20-30 mg of benzoin propionate and 2-3 L of dichloromethane.

5. The method for simultaneous detection of six chemicals in natural flavors according to claim 3 or 4, wherein The amount of the natural flavor is 10-200 mg, and the amount of the internal standard extractant is 25-50 mL.

6. The method for simultaneous detection of six chemicals in natural flavors according to claim 5, wherein, The amount of the natural flavor is 100-110 mg, and the amount of the internal standard extractant is 25-30 mL.

7. The method for simultaneous detection of six chemicals in natural flavors according to claim 1, wherein The mixing and extraction is further dried with anhydrous sodium sulfate.

8. The method for simultaneous detection of six chemicals in natural flavors according to claim 7, wherein The mass ratio of the anhydrous sodium sulfate to the natural flavor is 1:1-9.

9. The method for simultaneous detection of six chemicals in natural flavors according to claim 1 or 3 or 7 or 8, wherein The natural flavor is a tincture, an extract, an essential oil or a neat oil.

10. The method for simultaneous detection of six chemicals in natural flavors according to claim 1, wherein The gas chromatography-mass spectrometry analysis conditions are as follows: carrier gas: helium; column flow rate: 1 mL / min; injection port temperature: 250 DEG C; programmed temperature: 50 DEG C, 0 min, 5 DEG C / min→250 DEG C, 50 min, 250 DEG C for 10 min, then 260 DEG C for 10 min; split mode: no split; transfer line temperature: 250 DEG C; EI ion source temperature: 230 DEG C, quadrupole temperature: 150 DEG C; EI ionization energy: 70 eV; selected ion mode.

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