A method for simultaneous detection of 24 allergenic fragrances in perfume

Abstract

The present invention discloses a method for simultaneously detecting 24 allergenic fragrances in perfume. The method of the present invention is used to simultaneously detect 24 allergenic fragrances in perfume. The 24 allergenic fragrances show a good linear relationship in the range of 0.05 to 10 mg / L, and the detection limit is 0.5 to 25 μg / L. The method of the present invention is simple to operate, rapid, accurate, and highly sensitive, and the detection limit is significantly lower than the existing gas chromatography-mass spectrometry method, and is suitable for the simultaneous rapid determination of 24 allergenic fragrances in perfume.

Description

Technical Field

[0001] The invention relates to a method for simultaneously detecting 24 allergenic fragrances in perfume, belonging to the technical field of chemical detection. Background Art

[0002] Perfumes are mainly products composed of alcohol, water and spices. Spices can give perfumes a pleasant aroma, but they also have the risk of causing contact allergies. In recent years, the safety of daily-use spices has attracted widespread attention worldwide. In 2009, the EU Cosmetics Regulation stipulated that if the content of 26 allergenic spices in cosmetics (limonene, linalool, methyl 2-octynoate, citral, citronellol, geraniol, α-isomethyl ionone, benzyl alcohol, hydroxycitronellal, cinnamaldehyde, lily of the valley aldehyde, eugenol, α-amyl cinnamaldehyde, anise alcohol, cinnamyl alcohol, farnesol, isoeugenol, α-hexyl cinnamaldehyde, coumarin, lyral, α-amyl cinnamyl alcohol, benzyl benzoate, benzyl salicylate, benzyl cinnamate, oak moss extract and tree moss extract) must be marked on the cosmetics label. Among them, oak moss extract and tree moss extract are natural flavors. Since natural flavors are mixtures with complex ingredients and are affected by factors such as origin, production period, and preparation process, they are often analyzed separately.

[0003] The main detection methods for 24 allergenic synthetic fragrances in cosmetics include gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). However, since there are many aromatic substances in perfumes and their types are complex, with more than 100 peaks, there is a lot of interference when using conventional one-dimensional chromatographic detection such as gas chromatography and liquid chromatography. Sample chromatographic peaks usually overlap and baseline separation cannot be achieved. It is difficult to accurately determine the retention start and end time of the fragrance ingredients, which affects the accuracy of the qualitative and quantitative results.

[0004] Comprehensive two-dimensional gas chromatography combines two chromatographic columns with different separation mechanisms but independent of each other in series to form a two-dimensional gas chromatography. A modulator installed between the two chromatographic columns plays a role of capture and retransmission, and its peak capacity is the product of the peak capacities of the two chromatographic columns that make it up. Therefore, comprehensive two-dimensional gas chromatography provides a true orthogonal separation system with excellent separation ability, high peak capacity and high resolution. However, when comprehensive two-dimensional gas chromatography is currently used for the detection of allergenic fragrances in perfumes, it is still impossible to achieve the simultaneous detection of 24 allergenic fragrances. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for simultaneously and rapidly determining 24 allergenic fragrances in perfume, and specifically relates to a method for simultaneously and rapidly determining 24 allergenic synthetic fragrances in perfume using a comprehensive two-dimensional gas chromatography-high-resolution high-throughput time-of-flight mass spectrometer.

[0006] The object of the present invention is to provide a method for simultaneously detecting 24 allergenic fragrances in perfume, comprising the following steps:

[0007] S1. Prepare standard stock solutions of 24 allergenic fragrances using 70% to 90% ethanol by volume as solvent;

[0008] S2. Take the standard stock solutions of 24 allergenic fragrances and prepare mixed standard solutions with a concentration of 5 to 50 mg / L for each allergenic fragrance;

[0009] S3, using comprehensive two-dimensional gas chromatography-high-resolution high-throughput time-of-flight mass spectrometry to detect the mixed standard solution;

[0010] S4. Analyze the collected data and conduct quantitative analysis on the fragrance compounds.

[0011] Furthermore, the 24 allergenic fragrances include: limonene, linalool, methyl 2-octynoate, citral, citronellol, geraniol, α-isomethyl ionone, benzyl alcohol, hydroxycitronellal, cinnamaldehyde, lily of the valley aldehyde, eugenol, α-amyl cinnamaldehyde, anise alcohol, cinnamyl alcohol, farnesol, isoeugenol, α-hexyl cinnamaldehyde, coumarin, lyral, α-amyl cinnamyl alcohol, benzyl benzoate, benzyl salicylate and benzyl cinnamate.

[0012] Furthermore, in the mixed standard solution, the concentration of each allergenic fragrance is: 5 mg / L of α-amylcinnamyl alcohol, 20 mg / L of farnesol and isomethyl ionone, 30 mg / L of linalool and lily of the valley aldehyde, 40 mg / L of limonene, and the concentration of each remaining component is 10 mg / L.

[0013] Furthermore, the volume ratio concentration of the ethanol is 80%.

[0014] Furthermore, in the comprehensive two-dimensional gas chromatography-high-resolution high-throughput time-of-flight mass spectrometer, the one-dimensional chromatographic column is TR-FFAP and the two-dimensional chromatographic column is Rxi-17SilMS.

[0015] Furthermore, during the detection, the injection port temperature was 250° C., and the sample was injected in a splitless mode.

[0016] Furthermore, during the detection, the one-dimensional column oven temperature program was as follows: the initial temperature was 70°C and maintained for 5 min, then the temperature was increased to 240°C at a rate of 10°C / min and maintained for 5 min.

[0017] Furthermore, during the detection, the temperature raising program of the two-dimensional column oven was as follows: the starting temperature was 75°C and maintained for 5 min, then the temperature was raised to 245°C at a rate of 10°C / min and maintained for 5 min.

[0018] Furthermore, during the detection, high-purity helium was used as the carrier gas in a constant flow mode with a flow rate of 1 mL / min.

[0019] Furthermore, during detection, the time-of-flight mass spectrometry conditions were: using an EI ion source, an ion source temperature of 210°C, a transmission line temperature of 250°C, a voltage control of 70eV, a detector collection mass number range of 33 to 400amu, a collection frequency of 200spectra / s, and an electron bombardment energy of 2000V.

[0020] Furthermore, the collected data were analyzed using ChromaTOF software, and the peak widths of the first and second dimensions were set to 24 s and 0.2 s, respectively, and the fragrance compounds were quantitatively analyzed based on the mainlib database and mixed standards.

[0021] The beneficial effects of the present invention are:

[0022] The method of the present invention is used to simultaneously detect 24 allergenic fragrances in perfume. The 24 allergenic fragrances show a good linear relationship in the range of 0.05-10 mg / L, and the detection limit is 0.5-25 μg / L. The method of the present invention is simple to operate, rapid, accurate, and highly sensitive, and the detection limit is significantly lower than the existing gas chromatography-mass spectrometry, and is suitable for the simultaneous rapid determination of 24 allergenic fragrances in perfume. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 GC-MS graphs of mixed standard solutions (10 mg / L) prepared in ethanol (1) and methanol (2) respectively;

[0024] Figure 2 The GC-MS total ion flow diagram of 24 allergenic fragrance mixed standard solutions (10 mg / L) is shown in the figure. Among them, 1. limonene; 2. benzyl alcohol; 3. linalool; 4. 2-octynoic acid methyl ester; 5. citronellol; 6. geraniol; 7. cinnamaldehyde; 8. anise alcohol; 9. hydroxycitronellal; 10. cinnamyl alcohol; 11. citral; 12. eugenol; 13. coumarin; 14. isoeugenol; 15. α-isomethyl ionone; 16. lilyral; 17. α-amyl cinnamaldehyde; 18. lyral; 19. α-amyl cinnamyl alcohol; 20a and 20b, farnesol; 21. α-hexyl cinnamaldehyde; 22. benzyl benzoate; 23. benzyl salicylate; 24. benzyl cinnamate;

[0025] Figure 3 It is the GC×GC-TOFMS one-dimensional total ion chromatogram of 24 allergenic fragrances;

[0026] Figure 4 It is the GC×GC-TOFMS two-dimensional contour map of 24 allergenic fragrances;

[0027] Figure 5 It is the GC×GC-TOFMS three-dimensional image of 24 allergenic fragrances;

[0028] in, Figure 3 , 4 , 5, 1, limonene; 2, linalool; 3, methyl 2-octynoate; 4, citral; 5, citronellol; 6, geraniol; 7, α-isomethyl ionone; 8, benzyl alcohol; 9, hydroxycitronellal; 10, cinnamaldehyde; 11, lily of the valley aldehyde; 12, eugenol; 13, α-amyl cinnamaldehyde; 14, anise alcohol; 15, cinnamyl alcohol; 16a and 16b, farnesol; 17, isoeugenol; 18, α-hexyl cinnamaldehyde; 19, coumarin; 20, lyral; 21, amyl cinnamyl alcohol; 22, benzyl benzoate; 23, benzyl cinnamate; 24, benzyl salicylate. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0030] Example 1: Selection of solvent

[0031] The 24 allergenic fragrance compounds have a certain polarity. Methanol and ethanol were used as solvents to prepare mixed standard solutions with a concentration of 10 mg / L for each component for one-dimensional GC-MS detection. The specific test method is as follows.

[0032] GC conditions: TR-FFAP column (30m×0.25mmID, 0.25μm); programmed temperature: initial temperature 45°C, hold for 2min; increase to 245°C at 10°C / min, then increase to 280°C at 30°C / min, hold for 4min; carrier gas He (99.999%), constant flow mode, flow rate: 1.0mL / min; split ratio 1:10.

[0033] MS conditions: EI ion source, ion source temperature: 210°C, transfer line temperature: 280°C, voltage control 70 eV; mass scanning range: 33-400 amu.

[0034] The results showed that the peak response of each flavor compound when using ethanol was significantly higher than that of methanol. Figure 1 As shown, ethanol was selected as the solvent.

[0035] Example 2: GC-MS Analysis of Allergenic Flavors

[0036] A mixed standard solution with a concentration of 10 mg / L of each component was prepared using ethanol as the solvent for one-dimensional GC-MS detection. The specific test method is as follows.

[0037] GC conditions: TR-FFAP column (30m×0.25mmID, 0.25μm); programmed temperature: initial temperature 45°C, hold for 2min; increase to 245°C at 10°C / min, then increase to 280°C at 30°C / min, hold for 4min; carrier gas He (99.999%), constant flow mode, flow rate: 1.0mL / min; split ratio 1:10.

[0038] MS conditions: EI ion source, ion source temperature: 210°C, transfer line temperature: 280°C, voltage control 70 eV; mass scanning range: 33-400 amu.

[0039] The obtained GC-MS total ion current is as follows Figure 2 As shown. Figure 2 It can be clearly seen that some chromatographic peaks overlap, such as between compounds 8 and 9, between 13 and 14, and between 19 and 20(a), all of which have varying degrees of co-elution. The overlap between 8 and 9 is particularly serious, and generally can only be separated by manual retrieval, which will inevitably affect the accuracy of qualitative and quantitative results and make it difficult to obtain satisfactory results.

[0040] Example 3: GC×GC-TOFMS analysis of allergenic fragrances

[0041] According to the response peaks of the 24 spices in GC, the concentration of each spice in the mixed standard solution was re-determined. Using 80% ethanol as the solvent, a new mixed standard solution with a concentration of 5.0 mg / L of α-amylcinnamyl alcohol, 20 mg / L of farnesol and isomethyl ionone, 30 mg / L of linalool and lily of the valley aldehyde, 40 mg / L of limonene, and 10 mg / L of the remaining components was prepared. GC×GC-TOFMS was used to separate the mixed standard solution of 24 allergenic spices. The specific test method is as follows:

[0042] The conditions of comprehensive two-dimensional gas chromatography were as follows: one-dimensional column TR-FFAP (30 m × 0.25 mm ID, 0.25 μm), two-dimensional column Rxi-17SilMS (1.4 m × 0.25 mm ID, 0.25 μm), injection port temperature 250 °C, injection in splitless mode; one-dimensional column oven temperature program: starting temperature 70 °C for 5 min, heating to 240 °C at a rate of 10 °C / min and holding for 5 min; two-dimensional column oven temperature program The sequence is: the starting temperature is 75℃ and maintained for 5min, then the temperature is increased to 245℃ at 10℃ / min and maintained for 5min; high-purity helium is used as the carrier gas, constant flow mode, flow rate is 1mL / min; the time-of-flight mass spectrometry conditions are: using EI ion source, ion source temperature 210℃, transmission line temperature 250℃, voltage control 70eV, detector collection mass number range 33~400amu, collection frequency 200spectra / s, electron bombardment energy is 2000V. Data processing: The collected data are analyzed using the ChromaTOF software that comes with the instrument, and the peak widths of the first and second dimensions are set to 24s and 0.2s respectively. The fragrance compounds are quantitatively analyzed according to the mainlib database and mixed standards.

[0043] The total ion current chromatogram obtained is as follows Figure 3 As shown. Figure 3 It can be seen that although there are some overlapping chromatographic peaks in the one-dimensional total ion current, such as between geraniol (6) and α-isomethyl ionone (7), between cinnamaldehyde (10) and lily of the valley aldehyde (11), they are well separated in the two-dimensional. Figure 4 Two-dimensional iso-peak diagram and Figure 5 As shown in the three-dimensional diagram, it can be seen that GC×GC-TOFMS has high peak capacity and high resolution, and is significantly superior to ordinary one-dimensional GC-MS in the separation of 24 allergenic fragrance mixed standards.

[0044] The linear range, detection limit, quantification limit, accuracy and precision of the GC×GC-TOFMS method were evaluated. As shown in Table 1, the 24 allergenic fragrances have a good linear relationship in the concentration range of 0.05-10 mg / L, and the correlation coefficient R 2 >0.995. The detection limit of the method was in the range of 0.742-24.830 μg / L, and the quantification limit was in the range of 2.472-82.768 μg / L. The detection limit of the GC×GC-TOFMS method was lower and more sensitive.

[0045] Table 1 Linear equation, detection limit and quantification limit of GC×GC-TOFMS method

[0046]

[0047] Using 80% ethanol solution as blank matrix, three addition levels were set to 1.0, 5.0 and 10 mg / L, and each addition level was measured 5 times to calculate the spike recovery. The results are shown in Table 2. Except for benzyl cinnamate, the spike recovery rates at the three addition levels were in the range of 73% to 130%. At the 10 mg / L addition level, the relative standard deviations of 24 allergenic spices were 2.02% to 9.86% (n=5). The accuracy and precision of the method were good and could meet the analytical requirements.

[0048] Table 2 Blank matrix (80% ethanol) spike recovery

[0049]

[0050] Comparative Example 1:

[0051] Anhydrous ethanol was used as a solvent to prepare a mixed standard solution with a concentration of 5.0 mg / L of α-amylcinnamyl alcohol, 20 mg / L of farnesol and isomethyl ionone, 30 mg / L of linalool and lily of the valley aldehyde, 40 mg / L of limonene, and 10 mg / L of each of the other components. GC×GC-TOFMS was used to separate the mixed standard solutions of 24 allergenic fragrances, and the specific test method was the same as in Example 3.

[0052] The results showed that the concentrations of 24 allergenic fragrances had a good linear relationship in the range of 0.05-10 mg / L, and the correlation coefficient R 2 >0.990. In the mixed standard solution prepared above, three addition levels were set to 1.0, 5.0 and 10 mg / L, and each addition level was measured 5 times. The spiked recovery was calculated, and the results are shown in Table 3. As can be seen from Table 3, the number of spices with a recovery rate of 70% to 120% at the three addition levels was 10, 11 and 18 respectively. Therefore, the accuracy of this method does not meet the requirements.

[0053] Table 3 Recovery rates of 24 spices spiked

[0054]

[0055] Comparative Example 2:

[0056] A mixed standard solution with a concentration of 10 mg / L of each component was prepared using 80% ethanol as a solvent, and GC×GC-TOFMS was used to separate the mixed standard solution of 24 allergenic fragrances. The specific test method was the same as in Example 3.

[0057] The results showed that the concentrations of 24 allergenic fragrances had a good linear relationship in the range of 0.05-10 mg / L, and the correlation coefficient R 2>0.990. In the mixed standard solution prepared above, three addition levels were set to 1.0, 5.0 and 10 mg / L, and each addition level was measured 5 times. The spiked recovery rate was calculated. The results showed that only 11 spices had a recovery rate of 70% to 120% under the three addition levels, and the spiked recovery rates of the remaining spices were all beyond this range. Therefore, the accuracy of this method does not meet the requirements.

[0058] Example 4: Perfume Sample Detection

[0059] Pipette 1mL of 80% ethanol into a sample injection bottle, add 5μL of perfume sample, mix well, and obtain the sample solution to be tested. The established GC×GC-TOFMS method (Example 3) was used to determine the concentrations of 24 allergenic fragrances in three perfume samples (sample 1 female perfume, sample 2 baby perfume, sample 3 male perfume). The specific test method is as follows.

[0060] The conditions of comprehensive two-dimensional gas chromatography were as follows: one-dimensional column TR-FFAP (30 m × 0.25 mm ID, 0.25 μm), two-dimensional column Rxi-17SilMS (1.4 m × 0.25 mm ID, 0.25 μm), injection port temperature 250 °C, injection in splitless mode; one-dimensional column oven temperature program: starting temperature 70 °C for 5 min, heating to 240 °C at a rate of 10 °C / min and holding for 5 min; two-dimensional column oven temperature program The sequence is: the starting temperature is 75℃ and maintained for 5min, then the temperature is increased to 245℃ at 10℃ / min and maintained for 5min; high-purity helium is used as the carrier gas, constant flow mode, flow rate is 1mL / min; the time-of-flight mass spectrometry conditions are: using EI ion source, ion source temperature 210℃, transmission line temperature 250℃, voltage control 70eV, detector collection mass number range 33~400amu, collection frequency 200spectra / s, electron bombardment energy is 2000V. Data processing: The collected data are analyzed using the ChromaTOF software that comes with the instrument, and the peak widths of the first and second dimensions are set to 24s and 0.2s respectively. The fragrance compounds are quantitatively analyzed according to the mainlib database and mixed standards.

[0061] The results showed that a total of 16 allergenic fragrances were detected in the three perfume samples, of which 15 allergenic fragrances except isoeugenol exceeded the EU limit of 0.001%. However, all three perfumes were marked in the ingredient list and complied with the regulations.

[0062] Table 4 Detection results of 24 allergenic fragrances in three perfume samples

[0063]

[0064] *NF, Not Found.

[0065] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A method for simultaneously detecting 24 allergenic fragrances in perfume, characterized in that: The steps include: S1. Prepare standard stock solutions of 24 allergenic fragrances using 70% to 90% ethanol as solvent; S2. Take the standard stock solutions of 24 allergenic fragrances and prepare mixed standard solutions with the concentration of each allergenic fragrance ranging from 5 to 50 mg / L; S3, using comprehensive two-dimensional gas chromatography-high-resolution high-throughput time-of-flight mass spectrometry to detect the mixed standard solution; The first-dimensional column was TR-FFAP, the second-dimensional column was Rxi-17SilMS, the injection port temperature was 250 °C, and the injection was carried out in splitless mode; The temperature program of the first-dimensional column oven was as follows: the initial temperature was 70 °C and maintained for 5 min, then the temperature was increased to 240 °C at a rate of 10 °C / min and maintained for 5 min; The temperature program of the two-dimensional column oven was as follows: initial temperature 75°C, maintained for 5 min, increased to 245°C at 10°C / min and maintained for 5 min; High-purity helium was used as the carrier gas in constant flow mode with a flow rate of 1 mL / min; The EI ion source was used, the ion source temperature was 210°C, the transfer line temperature was 250°C, the voltage was controlled at 70 eV, the detector collected mass numbers in the range of 33-400 amu, the collection frequency was 200 spectra / s, and the electron bombardment energy was 2000 V; S4, analyzing the collected data and performing quantitative analysis on the fragrance compounds, wherein the steps of collecting data include transferring 1 mL of 80% ethanol into a sample injection bottle, adding 5 μL of the perfume sample to be tested, and mixing evenly to obtain a sample solution to be tested; The 24 allergenic fragrances include: limonene, linalool, methyl 2-octynoate, citral, citronellol, geraniol, α-isomethyl ionone, benzyl alcohol, hydroxycitronellal, cinnamaldehyde, lily of the valley aldehyde, eugenol, α-amyl cinnamaldehyde, anise alcohol, cinnamyl alcohol, farnesol, isoeugenol, α-hexyl cinnamaldehyde, coumarin, lily of the valley aldehyde, α-amyl cinnamyl alcohol, benzyl benzoate, benzyl salicylate and benzyl cinnamate.

2. The method according to claim 1, characterized in that: In the mixed standard solution, the concentration of each allergenic fragrance is: 5 mg / L of α-amylcinnamyl alcohol, 20 mg / L of farnesol and isomethyl ionone, 30 mg / L of linalool and lily of the valley aldehyde, 40 mg / L of limonene, and the concentration of each other component is 10 mg / L.

3. The method according to claim 1, characterized in that In step S4, the collected data were analyzed using ChromaTOF software, and the peak widths of the first and second dimensions were set to 24 s and 0.2 s, respectively. The fragrance compounds were quantitatively analyzed based on the mainlib database and mixed standards.

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