Method for detecting sensitization perfume neolilial in children cosmetics and application thereof

Abstract

The invention discloses a method for detecting sensitizing perfume neolilial in children cosmetics, which comprises the following steps: (1) adding a children cosmetic sample into a mixed solution consisting of formic acid, acetonitrile and water, and carrying out ultrasonic extraction to obtain a sample extract; (2) enabling the sample extract to pass through a molecularly imprinted solid-phase extraction column, and preparing a purified sample liquid to be detected by adopting gradient elution; (3) carrying out derivatization reaction on the purified sample liquid to be detected and a methylation reagent to obtain a derivatization product; and (4) introducing the derivatization product into a gas chromatograph-tandem mass spectrometer through an interface, and detecting the lilial. The method for detecting the neolilial in the children cosmetics, provided by the invention, has the advantages of high sensitivity, good selectivity, simplicity in operation and the like. The invention also discloses a system for detecting the sensitizing perfume neolilial in the children cosmetics. The system comprises a pretreatment module, a chromatographic separation module, a mass spectrometry detection module and a data analysis module.

Description

Technical Field

[0001] The present invention relates to the field of analysis and detection of children's cosmetics, and particularly to a method for detecting and applying the sensitizing fragrance lyral in children's cosmetics. Background Art

[0002] In today's society, the use of cosmetics is becoming increasingly popular, and the children's cosmetics market is also showing a growing trend. However, the safety issues of cosmetics, especially the sensitizing ingredients therein, have always been the focus of public concern. As one of the most frequently used and widely applied additives in cosmetics, the adverse reactions caused by fragrances are particularly prominent. According to relevant statistical data, at least 35% of cosmetic allergy cases are caused by fragrances, and this proportion is sufficient to attract our high attention to the safety of fragrances used in cosmetics (De G, Anton C, Frosch P J. Adverse reactions to fragrances: a clinical review[J]. Contact Dermatitis, 1997, 36: 57 - 86.).

[0003] As a common fragrance ingredient, lyral has a unique aroma quality, with a lasting lily - of - the - valley floral fragrance and a light, sweet and moist meadow - saffron flower fragrance, and is widely used in the formulation of various fragrance types of essence. It can be found in many perfume, soap and cosmetic products with a hundred - flower fragrance type. However, for such a widely used fragrance, its allergic problems have gradually emerged. A large number of test data and adverse reaction cases of cosmetic allergies provide strong evidence for the sensitization of lyral (SCCS OPINION on Fragrance allergens in cosmetic products Scientific Committee on Consumer Safety. 2012: 15th plenary meeting.). Based on this, on August 2, 2017, the European Commission officially issued Regulation (EU) 2017 / 1410, clearly adding lyral to the list of prohibited substances in Appendix II. Subsequently, the ASEAN Cosmetics Committee also officially issued a directive in January 2018 to prohibit the use of lyral in cosmetics. Nevertheless, the 2015 version of the "Technical Specifications for Cosmetics Safety" in China did not list lyral in the list of prohibited substances at that time, which to a certain extent reflects the differences and lag in China's cosmetics safety supervision compared with international standards, and also highlights the urgency of researching methods for detecting lyral.

[0004] In terms of detection technology, the current methods for determining the allergenic fragrance lyral in cosmetics mainly include gas chromatography, high performance liquid chromatography, etc. Although these methods can achieve quantitative detection of lyral to a certain extent, they all have their own shortcomings. For example, many detection methods have complex operation processes, long detection time, low sensitivity, or are easily interfered by other ingredients in cosmetics, which will affect the accuracy and reliability of the test results. These problems are already prominent in the detection of ordinary cosmetics, and in the special field of children's cosmetics, the problem is even more serious. The ingredients of children's cosmetics are often more complex, and considering the physiological and behavioral development characteristics of children, for example, children may have some behaviors such as sucking or scratching, which will increase their exposure to the ingredients in cosmetics. In addition, there are obvious differences between children's metabolic capacity and adults. These factors make the safety requirements of children's cosmetics much higher than those of ordinary cosmetics. Therefore, for children's cosmetics, we should use more rigorous evaluation data as much as possible to conduct safety assessments to ensure the health and safety of children.

[0005] However, the reality is that the existing detection technology means are far from meeting the needs of quality and safety supervision and technical guarantee for children's cosmetics. Taking the 2015 edition of my country's "Hygiene Standards for Cosmetics" as an example, although more than 1,000 banned and restricted substances are listed, there are only more than 300 substances with standardized inspection methods or standards. Specifically in the field of children's cosmetics, although domestic and foreign technical regulations such as the "Technical Standards for Safety of Cosmetics" have put forward prohibition or restriction requirements for fragrances, colorants, amides, pyrrolidones, halogenated hydrocarbon organic solvents, quaternary ammonium salt fungicides, etc. in cosmetics, there are still many problems to be solved in actual detection methods and regulatory measures. According to the new management requirements for children's cosmetics, one of the first problems to be solved is to establish a detection method for banned or restricted substances that may be used in children's cosmetics, including lyral, to fill the current method gaps and provide strong technical support and guarantee for the quality and safety supervision of children's cosmetics, so as to better protect children from potential harmful substances in cosmetics and safeguard children's health rights and interests. Therefore, it is very necessary to develop a detection method for lyral, an allergenic fragrance, in children's cosmetics. Summary of the invention

[0006] The purpose of the present invention is to provide a detection method and application of lyralin, an allergenic fragrance, in children's cosmetics.

[0007] In order to achieve the above object, the solution of the present invention is: A method for detecting lyral, an allergenic fragrance, in children's cosmetics comprises the following steps: (1) Sample pretreatment: Add the children's cosmetics sample into a mixed solution composed of formic acid, acetonitrile and water, and perform ultrasonic extraction to obtain a sample extract; (2) Purification treatment: Pass the sample extract prepared in step (1) through a molecularly imprinted solid-phase extraction column, and perform gradient elution to obtain a sample test solution after purification treatment; (3) Derivatization treatment: React the sample test solution after purification treatment obtained in step (2) with a methylation reagent to obtain a derivatization product; (4) GC-MS / MS detection: Introduce the derivatization product obtained in step (3) into a gas chromatography-tandem mass spectrometer through an interface for detection of helional, and the conditions of gas chromatography-tandem mass spectrometry are as follows: Capillary gas chromatography column: Use an HP-innowax capillary column or equivalent; Carrier gas: Use high-purity helium gas; Injection mode: Use split or splitless injection; Temperature programming mode: Use programmed temperature rise; The mass spectrometry parameters are as follows: Use a quadrupole mass spectrometer detector; Use an EI ion source.

[0008] Preferably, the mass fraction of formic acid in the mixed solution described in step (1) is 1%; the volume ratio of acetonitrile to water is 4:1.

[0009] Preferably, the specific conditions of the ultrasonic extraction described in step (1) are: the ultrasonic power is 300 - 500 W, the extraction temperature is 30 - 50 °C, and the extraction time is 10 - 20 min.

[0010] Preferably, the gradient elution program described in step (2) is as follows: 0 - 1.0 min: Maintain 5% methanol; 1.0 - 5.0 min: The methanol ratio linearly increases to 95%; 5.0 - 6.0 min: Maintain 95% methanol; 6.0 - 6.1 min: The methanol ratio drops to 5%; 6.1 - 8.0 min: Equilibrate the chromatographic column.

[0011] Preferably, the methylation reagent described in step (3) is one of methyl trifluoromethanesulfonate, iodomethane or dimethyl sulfate.

[0012] Preferably, the cleavage conditions described in step (3) are: the cleavage time is 10 - 45 s, and the cleavage temperature is 150 - 300 °C.

[0013] The detection method for the sensitizing fragrance Lyral in children's cosmetics further includes a quality control step, and the quality control step is as follows: blank control test, spiked recovery test, and parallel determination of quality control samples; among them, the spiking levels in the spiked recovery test are 10, 50, and 100 mg / kg respectively, and the RSD of the parallel determination of quality control samples ≤ 9%.

[0014] Preferably, in step (4), the energy of the EI ion source is 80 eV, and the ion source temperature is 220 °C.

[0015] A detection system for the sensitizing fragrance Lyral in children's cosmetics includes: A pretreatment module configured with a temperature-controlled ultrasonic extraction device and an automatic solid-phase extraction workstation; A chromatographic separation module including a gas chromatograph and a constant-temperature column oven; A mass spectrometry detection module using a triple quadrupole mass spectrometer; A data analysis module with a matrix interference recognition system based on machine learning algorithms built therein.

[0016] The application of the detection method for the sensitizing fragrance Lyral in children's cosmetics described above, and the application is applicable to children's cosmetics in different dosage forms such as creams, powders, and liquids.

[0017] The principle of the detection method for the sensitizing fragrance Lyral in children's cosmetics provided by the present invention is as follows: The detection method for the sensitizing fragrance Lyral in children's cosmetics provided by the present invention is based on GC-MS / MS detection; however, due to the characteristics of complex matrix composition and similar structures of the detection target substances in children's cosmetics; effective pretreatment is required before detection. On the one hand, this can effectively reduce the interference of complex base substances in the cosmetic samples on the GC-MS / MS test, and on the other hand, it can also effectively concentrate the contained Lyral, thereby improving the accuracy of the analysis and test. Therefore, the method provided by the present invention applies the molecular imprinting technology to the pretreatment of samples; specifically, in the present invention, Lyral is used as the template of the "imprinted" target molecule in the polymer in advance to form highly specific binding sites, thereby realizing the selective adsorption and separation of the target molecule. During the sample treatment using the polymer molecular imprinting technology, the molecularly imprinted solid-phase extraction column can enrich the target molecule Lyral from the complex cosmetic sample matrix to the surface of the polymer. Due to the enrichment effect, the concentration of the target molecule increases significantly in the extraction column, so that a stronger signal can be generated in the subsequent detection; this enrichment effect is particularly obvious in samples with low target molecule content and can significantly improve the sensitivity of the GC-MS / MS test.

[0018] The advantages of the detection method for the sensitizing fragrance Lyral in children's cosmetics provided by the present invention are as follows: (1) The detection method of lyral in children's cosmetics provided by the present invention has the advantages of high sensitivity, good selectivity and simple operation.

[0019] (2) The detection method of lyral in children's cosmetics provided by the present invention can be successfully applied to the detection of the content of lyral in a variety of children's cosmetics.

[0020] (3) The detection method of lyral in children's cosmetics provided by the present invention successfully applies the molecular imprinting technology to the detection of the content of lyral in children's cosmetics, opening up a new idea for the detection of lyral in complex matrices. Specific embodiments

[0021] The present invention will be further described in detail below in conjunction with embodiments. It should also be understood that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the protection scope of the present invention. The specific quality, reaction time and temperature, process parameters, etc. in the examples are also only an example within a suitable range. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention fall within the protection scope of the present invention.

[0022] Unless otherwise specified, the reagents used are commercial reagents and have not been further purified before use.

[0023] The samples used in the embodiments of the present invention are artificial simulated positive samples because the content of lyral in commercially available children's cosmetic samples is different, and the actual samples are not suitable for detecting and analyzing the content of lyral in children's cosmetics. Therefore, the specific preparation process of the positive children's cosmetic sample used in the embodiments of the present invention is as follows: Add lyral to 5 g of a children's cosmetic sample (baby moisturizing cream) with a negative detection result to ensure that the added amount of lyral in the positive sample is 40 mg / kg to obtain a test solution of a children's cosmetic containing lyral. It should be noted here that: there are two isomers of lyral: lyral Ⅰ CAS 31906-04-4 and lyral Ⅱ CAS 51414-25-6; in the embodiments of the present invention, lyral Ⅰ CAS 31906-04-4 is used as an example for detection, so the lyral in the present invention all refers to lyral Ⅰ.

[0024] The preparation steps of the lyral-methylacrylic acid-ethylene glycol dimethacrylate polymer molecular imprinting material used in the embodiments of the present invention are as follows: Under a nitrogen atmosphere, 0.1 g of benzoyl peroxide, 0.3 g of hydroxycitronellal, 1.2 g of methacrylic acid, and 6 g of ethylene glycol dimethacrylate were added to 100 ml of acetonitrile. After the resulting mixture was stirred evenly, the thermal polymerization was carried out at 60 °C for 24 hours, and then the reaction was stopped. It was naturally cooled to room temperature. The obtained solid was washed 3 times with 10 ml of acetonitrile, and then placed in a solution of 25 ml of methanol / acetic acid (9:1, v / v) and stirred at room temperature for 24 hours to elute the template molecule hydroxycitronellal. The polymer after removing the template molecule hydroxycitronellal was washed 3 times with 10 ml of acetonitrile again. Finally, the obtained solid was dried overnight in a vacuum drying oven at 80 °C. The dried polymer was ground and screened through a 200-mesh sample sieve. The obtained molecularly imprinted polymer material was denoted as MAA-EGDMA-1. According to the same preparation steps, the dosage of hydroxycitronellal was adjusted to 0.1 g, 0.6 g, and 0 g in sequence to prepare molecularly imprinted polymer materials MAA-EGDMA-2, MAA-EGDMA-3, and MAA-EGDMA-4 respectively. The prepared molecularly imprinted polymer materials were filled into solid-phase extraction columns to obtain molecularly imprinted solid-phase extraction columns.

[0025] Example 1 The steps for the method for detecting hydroxycitronellal in children's cosmetics are as follows: (1) Sample pretreatment: 1 g of the sample solution to be tested of children's cosmetics containing hydroxycitronellal was added to a mixed solution composed of acetonitrile and water (volume ratio 4:1) with a mass fraction of formic acid of 1%. The resulting mixture was ultrasonically extracted for 15 min under the conditions of an ultrasonic power of 300 W and an extraction temperature of 40 °C to obtain a sample extract. (2) Purification treatment: The sample extract prepared in step (1) was passed through a molecularly imprinted solid-phase extraction column filled with MAA-EGDMA-1 polymer material (column diameter 0.5 cm, packing length 5 cm), and then gradient elution was adopted. After combining the obtained eluates, a sample solution to be tested after purification treatment was obtained. Among them, the gradient elution program is as follows: 0 - 1.0 min: Maintain 5% methanol; 1.0 - 5.0 min: The methanol ratio linearly increases to 95%; 5.0 - 6.0 min: Maintain 95% methanol; 6.0 - 6.1 min: The methanol ratio drops to 5%; 6.1 - 8.0 min: Equilibrate the chromatographic column; (3)The sample test solution after purification obtained in step (2) is dried with nitrogen at 40 °C. The obtained residue is dissolved in 1.0 ml of acetone and then filtered through a 0.22 µm filter membrane. 5 microliters of an acetone solution containing 25% methyl trifluoromethanesulfonate is added to the obtained filtrate, and then the obtained mixture is loaded into a pyrolyzer for pyrolysis; pyrolysis conditions: pyrolysis temperature 200 °C, time 25 s, to obtain a derivatized product; (4)The derivatized product obtained in step (3) is introduced into a gas chromatography-tandem mass spectrometry (GC-MS / MS) through an interface for detection and analysis; GC-MS / MS conditions: the chromatographic column is HP-innowax; temperature programming: the initial column temperature is maintained at 60 °C for 2 min, heated to 300 °C at a rate of 15 °C / min, and maintained for 6 min. The temperature of the quadrupole is set at 150 °C, the energy of the EI ion source is 80 eV, and the ion source temperature is 220 °C. Each sample is measured in parallel 5 times, and the test result is: the content of lyral is 47 mg / kg.

[0026] The steps for the recovery and repeatability tests are as follows: A standard addition recovery test is carried out on a baby moisturizer detected as negative. The standard addition levels of lyral are 10, 50, and 100 mg / kg respectively, and the test conditions are the same as above; the results are as follows: when the standard addition level is 10 mg / kg, the recovery rate is 95% and the RSD is 4%; when the standard addition level is 50 mg / kg, the recovery rate is 99% and the RSD is 5%; when the standard addition level is 100 mg / kg, the recovery rate is 103% and the RSD is 8%.

[0027] Example 2 The steps of the method for detecting lyral in children's cosmetics refer to Example 1, except that the MAA-EGDMA-1 polymer material in step (2) is replaced with the MAA-EGDMA-2 polymer material; the remaining steps are the same as in Example 1, and the test result is: the content of lyral is 44 mg / kg.

[0028] Example 3 The steps of the method for detecting lyral in children's cosmetics refer to Example 1, except that the MAA-EGDMA-1 polymer material in step (2) is replaced with the MAA-EGDMA-3 polymer material; the remaining steps are the same as in Example 1, and the test result is: the content of lyral is 45 mg / kg.

[0029] Example 4 The steps of the method for detecting lyral in children's cosmetics refer to Example 1, except that the MAA-EGDMA-1 polymer material in step (2) is replaced with MAA-EGDMA-4 polymer material; the remaining steps are the same as those in Example 1, and the test result is: the content of lyral is 38 mg / kg.

[0030] Example 5 The steps of the method for detecting lyral in children's cosmetics refer to Example 1, except that the specific steps of step (2) are changed to: passing the sample extract obtained in step (1) through a molecularly imprinted solid phase extraction column filled with MAA-EGDMA-1 polymer material, and then washing the solid phase extraction column with 10 ml of 95% methanol, and combining the obtained liquid to obtain the sample test solution after purification treatment; the remaining steps are the same as those in Example 1, and the test result is: the content of lyral is 42 mg / kg.

[0031] Example 6 The present invention also provides a detection system for sensitizing fragrance lyral in children's cosmetics, including: A pretreatment module equipped with a temperature-controlled ultrasonic extraction device and an automatic solid phase extraction workstation; wherein, the pretreatment module may further include an intelligent weighing unit with an accuracy of 0.0001 g; and a multi-channel pipetting device supporting accurate pipetting in the range of 8 - 1000 μL. A chromatographic separation module including a gas chromatograph and a constant temperature column oven; wherein, the chromatographic separation module further includes a binary high-pressure gradient pump with a pressure resistance range of 0 - 1200 bar; an automatic sampler supporting a 96-position sample tray and temperature control function; the temperature control accuracy of the constant temperature column oven is ±0.5 °C, and the temperature range is 5 - 80 °C. A mass spectrometry detection module using a triple quadrupole mass spectrometer; wherein, the mass spectrometry detection module includes: a dynamic background subtraction system for real-time elimination of matrix interference; a mass axis calibration unit automatically calibrated with perfluorotributylamine; a data acquisition system with a sampling frequency of up to 50 Hz. A data analysis module with a matrix interference recognition system based on machine learning algorithms built-in; wherein, the data analysis module includes: an automatic integration algorithm capable of identifying peak shape distortion and correction; a homolog screening unit based on a retention time - mass-to-charge ratio two-dimensional database; an uncertainty evaluation unit automatically calculating the expanded uncertainty (k = 2).

[0032] Comparative Example 1 The steps of the method for detecting lyral in children's cosmetics refer to Example 1, except that the MAA-EGDMA-1 polymer material in step (2) is replaced with commercially available 996 silica gel material; the remaining steps are the same as those in Example 1, and the test result is: the content of lyral is 27 mg / kg.

[0033] Comparative Example 2 The steps of the method for detecting methylheptine carbonate in children's cosmetics refer to Example 1, except that the MAA-EGDMA-1 polymer material in step (2) is replaced with a commercially available neutral alumina material; the remaining steps are the same as in Example 1, and the test result is: the content of methylheptine carbonate is 26 mg / kg.

Claims

1. A method for detecting Lyral, an allergenic fragrance in children's cosmetics, characterized in that It includes the following steps: (1) Sample pretreatment: Add the children's cosmetic sample into a mixed solution composed of formic acid, acetonitrile and water, and perform ultrasonic extraction to obtain a sample extract; (2) Purification treatment: Pass the sample extract obtained in step (1) through a molecularly imprinted solid phase extraction column, and perform gradient elution to obtain a sample to be tested after purification treatment; (3) Derivatization treatment: Perform a derivatization reaction on the sample to be tested after purification treatment obtained in step (2) with a methylation reagent to obtain a derivatization product; (4) GC-MS / MS detection: Import the derivatization product obtained in step (3) into a gas chromatography-tandem mass spectrometer through an interface for the detection of lyral, and the conditions of gas chromatography-tandem mass spectrometry are as follows: Capillary gas chromatography column: Use an HP-innowax capillary column or an equivalent; Carrier gas: Use high-purity helium gas; Injection mode: Use split or splitless injection; Temperature programming mode: Use temperature programming; The mass spectrometry parameters are as follows: Use a quadrupole mass detector; Use an EI ion source.

2. The detection method of the sensitizing fragrance Lyral in children's cosmetics according to claim 1, characterized in that, In the mixed solution described in step (1), the mass fraction of formic acid is 1%; the volume ratio of acetonitrile to water is 4:

1.

3. The detection method of lyral as an allergenic fragrance in children's cosmetics according to claim 1, characterized in that The specific conditions of the ultrasonic extraction described in step (1) are: the ultrasonic power is 300 - 500 W, the extraction temperature is 30 - 50 °C, and the extraction time is 10 - 20 min.

4. The detection method of lilial, an allergenic fragrance in children's cosmetics according to claim 1, is characterized in that, The gradient elution program described in step (2) is as follows: 0 - 1.0 min: Keep 5% methanol; 1.0 - 5.0 min: The proportion of methanol linearly increases to 95%; 5.0 - 6.0 min: Keep 95% methanol; 6.0 - 6.1 min: The proportion of methanol drops to 5%; 6.1 - 8.0 min: Equilibrate the chromatographic column.

5. The detection method of the sensitizing fragrance Lyral in children's cosmetics according to claim 1, characterized in that, The methylation reagent described in step (3) is one of methyl trifluoromethanesulfonate, methyl iodide or dimethyl sulfate.

6. The detection method of lyral, an allergenic fragrance in children's cosmetics according to claim 1, is characterized in that, The cleavage conditions described in step (3) are: the cleavage time is 10 - 45 s, and the cleavage temperature is 150 - 300 °C.

7. The detection method of lyral, an allergenic fragrance in children's cosmetics according to claim 1, is characterized in that It also includes a quality control step, and the quality control step is as follows: blank control test, spiked recovery test and parallel determination of quality control samples; among them, the spiked levels in the spiked recovery test are 10, 50 and 100 mg / kg respectively, and the RSD of the parallel determination of quality control samples ≤ 9%.

8. The detection method of lyral, an allergenic fragrance in children's cosmetics according to claim 1, is characterized in that, The energy of the EI ion source described in step (4) is 80 ev, and the ion source temperature is 220 °C.

9. A detection system for lyral, an allergenic fragrance in children's cosmetics, characterized in that, It includes: A pretreatment module equipped with a temperature-controlled ultrasonic extraction device and an automatic solid phase extraction workstation; A chromatographic separation module containing a gas chromatograph and a constant temperature column oven; A mass spectrometry detection module using a triple quadrupole mass spectrometer; A data analysis module with a matrix interference recognition system based on a machine learning algorithm built-in.

10. The application of the method for detecting lyral in children's cosmetics according to claims 1 - 8 or the detection system according to claim 9, and the application is applicable to children's cosmetics of different dosage forms such as creams, powders and liquids.