Determination method for real release amount of volatile substances in mosquito repellent patch

Abstract

The invention discloses a method for measuring the real release amount of volatile substances in a mosquito repellent patch, which comprises the following steps: (I) placing an experiment cabin in a constant temperature box, placing a sample in the experiment cabin, simulating the natural environment, and sampling after balancing; and (II) measuring the migration release amount of volatile matters in the mosquito repellent patch by adopting a gas chromatography-mass spectrometry method. The invention also provides a method for measuring the content of volatile substances in the mosquito repellent patch and a method for screening a positive sample of the mosquito repellent patch. The method for detecting the real release amount of the risky substances in the novel mosquito-repelling product has important significance on effectively protecting consumers to establish related technical laws and regulations and limit standards of the novel mosquito-repelling product.

Description

Technical Field

[0001] This invention relates to the field of inspection and quarantine, and in particular to a method for determining the actual release amount of volatile substances in mosquito repellent patches. Background Technology

[0002] Mosquito bites cause skin redness, itching, and dermatitis, and are vectors for many infectious diseases such as malaria and dengue fever, not only affecting normal life but also seriously threatening people's health. Therefore, mosquito repellent products have become an increasingly popular choice. In recent years, with the increasing awareness of health, in addition to traditional mosquito repellent products such as mosquito coils, aerosol insecticides, and electric mosquito repellent liquids, new mosquito repellent products made with natural plant essential oils, such as mosquito repellent rings, patches, clips, and liquids, are widely popular due to their naturalness and lack of pesticide additives, and are used by special groups such as children and pregnant women. These plant-based mosquito repellent products (mosquito repellent patches, bracelets, and clips) are still relatively new. Currently, countries such as the United States, Canada, Australia, and Japan have established relevant regulations on the age and content of mosquito repellent products containing DEET, but have not made relevant regulations on potentially hazardous compounds such as toluene and other allergenic aromatics. However, my country's national standard "General Technical Conditions for Safety of Household Hygiene Insecticides" (GB 24330-2020) only applies to mosquito coils, sprays, and other insecticide and mosquito-repellent products, and lacks technical specifications and testing methods adapted to new plant-derived mosquito repellent products. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for determining the actual release amount of volatile substances in mosquito repellent patches that is low in cost and easy to operate.

[0004] A method for determining the content of volatile substances in mosquito repellent patches includes the following steps: sample pretreatment followed by analysis, under the following conditions:

[0005] Chromatographic conditions: HP-5MS column, 30m×0.25mm×0.25μm; injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃, hold for 1 min, increase to 160℃ at 15℃ / min, then increase to 280℃ at 20℃ / min, hold for 5 min;

[0006] Mass spectrometry conditions: EI ionization mode, transfer line temperature 280℃; ion source temperature 230℃; quadrupole temperature 150℃; solvent delay 3.0 min; selected ion monitoring mode.

[0007] The method for determining the content of volatile substances in the mosquito repellent patch of the present invention, wherein the volatile substances are α-pinene, D-limonene, and eucalyptol, and the chromatographic and mass spectrometric analysis parameters are as follows:

[0008]

[0009] Note: * indicates quantitative ions.

[0010] The method for determining the content of volatile substances in mosquito repellent patches according to the present invention further includes sample pretreatment: take 0.1g of a mosquito repellent patch, cut it into 3mm×3mm fragments, put it into a 10mL small brown bottle, add 5mL of dichloromethane, seal tightly, and extract by ultrasonication at room temperature for 30min. After ultrasonication, place it at room temperature and then filter it through a membrane for analysis.

[0011] A method for determining the actual release amount of volatile substances in mosquito repellent patches includes the following steps:

[0012] (I) Place the experimental chamber in a constant temperature chamber, and place the sample in the experimental chamber to simulate the natural environment. Take the sample after equilibration.

[0013] (II) The migration and release of volatile substances in the mosquito repellent patch were determined by gas chromatography-mass spectrometry under the following analytical conditions:

[0014] Thermal desorption conditions: sampling tube is Tenax TA, TDU temperature is 300℃, desorption time is 7min, cold trap CIS temperature is -10℃, and collection time is 8min;

[0015] Chromatographic conditions: HP-5MS column, 30m×0.25mm×0.25μm; injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃, hold for 1min, increase to 160℃ at 15℃ / min, then increase to 280℃ at 20℃ / min, hold for 5min.

[0016] Mass spectrometry conditions: ionization mode was EI, transfer line temperature was 280℃; ion source temperature was 230℃; quadrupole temperature was 150℃; solvent delay was 3.0 min; and the monitoring mode was selected ion monitoring (SIM).

[0017] The method for determining the actual release amount of volatile substances in the mosquito repellent patch of the present invention, wherein the volatile substances are α-pinene, D-limonene, and eucalyptol, and the chromatographic and mass spectrometric analysis parameters are as follows:

[0018]

[0019] Note: * indicates quantitative ions.

[0020] The method for determining the actual release amount of volatile substances in the mosquito repellent patch described in this invention includes:

[0021] The experimental chamber includes a chamber body and a cover covering the upper part of the chamber body. A fan is installed in the lower part of the chamber body and the fan is connected to an external power source through a wire. Multiple sample tray supports are fixed in the middle of the chamber body and grid-shaped sample trays are placed on them. An air inlet pipe is connected to the lower part of the chamber body, with its lower end connected to the internal space of the chamber body and its upper end connected to an air source through the air inlet pipe.

[0022] An air outlet and two sampling ports are provided on the outside of the cover. The air outlet is connected to a filter unit, and a sealing cover is detachably provided on the sampling ports. A temperature sensor is provided on the inside of the cover and is connected to an external power source via a wire.

[0023] The method for determining the actual release amount of volatile substances in mosquito repellent patches according to the present invention includes an experimental chamber that further includes a sampling tube. During the sampling process, the sealing cover is removed, and the sampling tube and the sampling port are connected by a connector. The chamber and the cover are connected by an adjustable sealing fastener. A pressure divider valve and a flow meter are provided on the air inlet pipe.

[0024] The method for determining the actual release amount of volatile substances in the mosquito repellent patch of the present invention, wherein step (I) specifically includes the following steps:

[0025] The experimental chamber was placed in a pre-set temperature and humidity chamber (26℃, 50% humidity). The chamber fan was turned on, and the air circuit control switch was activated. The air intake flow rate was controlled at 500 mL / min via the pressure divider valve and flow meter, allowing air to enter the chamber at a flow rate of 500 mL / min. The fan facilitated rapid diffusion of the gas within the chamber, which was then expelled through the outlet. After equilibration for 1 hour, the chamber lid was opened, and four mosquito repellent patches were placed on the sample tray. The lid was then replaced, and the sealing fasteners and cap were tightened. After the patches were placed in the chamber for equilibration for 0.5 hours, the volatile substances in the patches were collected after different release times were selected as needed. Two aged Tenax tubes were used for collection. The TA thermal desorption tubes were used as sampling tubes. The inlet ends of the two thermal desorption tubes were connected to the sampling port via connectors, and the outlet ends were connected to the sampling pump via silicone tubes. The sampling pump was set to a sampling flow rate of 150 mL / min and a sampling time of 10 min. Under this program, 1500 mL of gas was collected from the experimental chamber. After sampling, the sampling tubes were removed. The thermal desorption tubes with collected samples were placed on the sample tray of the thermal desorption-gas chromatography-mass spectrometry (TCMA) instrument, and the experimental parameters were set for qualitative and quantitative analysis.

[0026] A method for screening positive samples from mosquito repellent patches, comprising the following steps:

[0027] (A) Sample pretreatment: Crush the sample into particles smaller than 5 mm, weigh 0.1 g into a 20 mL headspace vial, seal it, and place it into a headspace sampler;

[0028] (B) Analysis conditions:

[0029] Thermal desorption conditions: sampling tube is Tenax TA, TDU temperature is 300℃, desorption time is 7min, cold trap CIS temperature is -10℃, and collection time is 8min;

[0030] Chromatographic conditions: HP-5MS column, 30m×0.25mm×0.25μm; injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃, hold for 1 min, increase to 160℃ at 15℃ / min, then increase to 280℃ at 20℃ / min, hold for 5 min;

[0031] Mass spectrometry conditions: ionization mode: EI; transfer line temperature: 280℃; ion source temperature: 230℃; quadrupole temperature: 150℃; monitoring mode: full scan monitoring; scan range: 40-500 m / z.

[0032] Headspace conditions: equilibrium temperature 60℃, equilibrium time 30 min, quantitative loop temperature 100℃, transfer line temperature 120℃, quantitative loop volume 1 mL, injection time 30 s;

[0033] (C) Screening positive samples: After obtaining the analytical structure, the samples containing α-pinene, D-limonene and eucalyptol were sorted by peak area, and the samples in which the peak areas of all three substances were above the median value were selected as positive samples.

[0034] The method for determining the content of volatile substances in mosquito repellent patches according to the present invention, or the method for establishing the method for determining the actual release amount of volatile substances in mosquito repellent patches according to the present invention, is characterized by comprising the following steps:

[0035] (a) Preparation of standard solutions;

[0036] (b) Screening positive samples from mosquito repellent patches;

[0037] (c) Optimize various experimental parameters.

[0038] The method for determining the actual release amount of volatile substances in the mosquito repellent patch of this invention differs from existing technologies in that:

[0039] Ingestion of fragrances may cause liver and kidney toxicity and endocrine disorders. Exposure to allergenic fragrances in certain populations may lead to allergic skin inflammations such as urticaria and itching. Excessive inhalation can cause sensory irritation such as burning and stinging in the eyes and nose, as well as asthma reactions. Therefore, researching methods for detecting the actual release levels of risk substances in novel mosquito repellent products is crucial for effectively protecting consumers and establishing relevant technical regulations and limits for these products. This invention, using widely used mosquito repellent patches as an example, employs a small-scale experimental chamber to simulate real-world usage scenarios and combines this with a thermal desorption technology testing method to provide scientific support for the risk assessment of novel mosquito repellent products.

[0040] The method for determining the actual release amount of volatile substances in the mosquito repellent patch of the present invention will be further explained below with reference to the accompanying drawings. Attached Figure Description

[0041] Figure 1 This is a graph showing the detection of 15 high-risk substances in 23 mosquito repellent patches using the method of this invention.

[0042] Figure 2 This is a diagram showing the optimized results of the desorption tube in the method of this invention;

[0043] Figure 3 The diagram shows the optimized results of TDU and CIS temperatures in the method of this invention.

[0044] Figure 4 This is a schematic diagram of the experimental chamber in this invention (wherein, the fan 3, sample tray support 4, sample tray 5 and temperature sensor 10 are inside the chamber 1). Detailed Implementation

[0045] I. A method for screening positive samples from mosquito repellent patches

[0046] Most substances are highly volatile. If traditional methods are used to prepare positive samples by adding spikes, most volatile substances are likely to be lost during the preparation process, making it difficult to guarantee the study of migration patterns and migration models for high-risk substances. Besides preparing self-made positive samples, actual samples can be used. Therefore, it is necessary to screen a large number of actual samples for target analytes to select positive samples with high detection quantities and concentrations of target analytes. In this invention, headspace chromatography-mass spectrometry is simple and quick to operate; an appropriate amount of sample can be directly placed in a headspace vial for analysis, making it a rapid screening method for volatile substances. Gas chromatography-mass spectrometry has high sensitivity and is further aided by the National Institute of Standards and Technology (NIST) mass spectral library. Therefore, this invention uses 23 mosquito repellent patches as the research object, employing headspace-gas chromatography / mass spectrometry to rapidly screen for the presence of volatile substances in novel mosquito repellent patches, in order to select positive samples for subsequent research.

[0047] 1.1 Experimental Section

[0048] (1) Instruments and Materials

[0049] 6890-5975 gas chromatograph-mass spectrometer (Agilent Technologies, USA); HSS 86.50 headspace sampler (DANI, Italy).

[0050] The mosquito repellent patch samples from different brands were all purchased from online e-commerce platforms and stored in sealed bags before the experiment to avoid cross-contamination.

[0051] (2) Sample pretreatment

[0052] The sample was crushed into particles smaller than 5 mm, 0.1 g was weighed into a 20 mL headspace vial, sealed, and placed into a headspace sampler.

[0053] (3) Instrument conditions

[0054] Chromatographic conditions: HP-5MS column (30m×0.25mm×0.25μm); injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃ held for 1min, increased to 160℃ at 15℃ / min, then increased to 280℃ at 20℃ / min, held for 5min.

[0055] Mass spectrometry conditions: ionization mode was EI, transfer line temperature was 280℃; ion source temperature was 230℃; quadrupole temperature was 150℃; monitoring mode was full scan monitoring, and the scan range was 40-500 m / z.

[0056] Headspace conditions: equilibrium temperature 60℃, equilibrium time 30 min, quantitative loop temperature 100℃, transfer line temperature 120℃, quantitative loop volume 1 mL, injection time 30 s.

[0057] 1.2 Results and Discussion

[0058] (1) Optimization of method conditions

[0059] Based on the top 15 high-risk substances identified in the preliminary non-targeted screening of new mosquito repellent products such as mosquito repellent bracelets, clips, and patches, as shown in Table 1, it can be seen that all substances in the table are volatile compounds. Since headspace testing for volatile compounds has the advantage of high sensitivity, and is simple and rapid, it can be used as the preferred method for rapid screening of volatile compounds. Gas chromatography-mass spectrometry also has high sensitivity for volatile compounds and is equipped with the NIST spectral library, giving it a significant advantage in screening and identifying volatile compounds. Therefore, this method uses headspace combined with gas chromatography-mass spectrometry as the screening method for positive samples from mosquito repellent patches in the preliminary screening.

[0060] Table 1. List of potential stress-generating substances in mosquito repellent products

[0061]

[0062] Considering the polarity of each substance, this study selected the moderately polar HP-5MS column for separation. The injection port temperature, split ratio, and temperature program were optimized to determine the optimal analytical conditions for chromatographic mass spectrometry. The headspace equilibration temperature and time were set at 60℃ for 30 min, which generally meets the sensitivity requirements for analyzing small-molecule volatile compounds. Since this method is primarily used to screen positive samples, only semi-qualitative analysis using the NIST library is required; therefore, there is no need to investigate linear range, recovery rate, etc.

[0063] (2) Sample screening results

[0064] Based on the 15 high-risk substances in mosquito repellent products shown in Table 1, this study used a headspace gas chromatography / mass spectrometry method to screen 23 mosquito repellent patches of different brands purchased from online e-commerce platforms for high-risk substances. The detection status of the 15 high-risk substances in the 23 mosquito repellent patches is as follows: Figure 1 As shown. From Figure 1 It can be seen that among the 23 mosquito repellent patches, the substances with relatively high detection rates and quantities are D-limonene (ranked 2nd), α-pinene (ranked 15th), eucalyptol (ranked 3rd), toluene (ranked 4th), and 1,4-cineole (ranked 8th). Therefore, these three substances can be selected as representative of volatile substances in mosquito repellent patches. Then, the samples containing all three substances were sorted by peak area. Samples with peak areas of all three substances above the median value were selected as positive samples. Sample 7 met the requirements for peak areas of D-limonene, α-pinene, and eucalyptol, and was therefore selected as a positive sample for subsequent research.

[0065] II. Research on the determination method of volatile substances in mosquito repellent patches (method establishment)

[0066] 2.1 Experimental Section

[0067] (1) Instruments, reagents and materials

[0068] 6890-5975 Gas Chromatograph-Mass Spectrometer (Agilent Technologies, USA); P300H Ultrasonic Cleaner (Elma GmbH, Germany).

[0069] α-pinene, D-limonene, eucalyptol and other standards were purchased from Shanghai Anpu Experimental Technology Co., Ltd., with a purity ≥98%; dichloromethane was chromatographically pure and purchased from Fisher Scientific, USA; nylon 66 filter membrane (0.22μm, Tianjin Jinteng Co., Ltd.)

[0070] (2) Preparation of standard solutions

[0071] Weigh 20 mg (accurate to 0.0001 g) of each standard into a 10 mL volumetric flask, dissolve and dilute to volume with dichloromethane to prepare a single-standard stock solution with a mass concentration of 2000 mg / L. Store in a brown stock bottle (protected from light at 4°C). Prepare a mixed stock solution with a mass concentration of 100 mg / L using dichloromethane. Dilute with dichloromethane to the appropriate concentration as needed before use.

[0072] (3) Sample pretreatment for content determination

[0073] After screening positive samples, take 0.1g of a mosquito repellent patch, cut it into 3mm×3mm fragments, put it into a 10mL small brown bottle, add 5mL of dichloromethane, seal tightly, and extract by ultrasonication at room temperature for 30min. After ultrasonication, place it at room temperature and then filter it through a membrane for analysis.

[0074] (4) Instrument conditions

[0075] Chromatographic conditions: HP-5MS column (30m×0.25mm×0.25μm); injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃ held for 1min, increased to 160℃ at 15℃ / min, then increased to 280℃ at 20℃ / min, held for 5min.

[0076] Mass spectrometry conditions: EI ionization mode, transfer line temperature 280℃; ion source temperature 230℃; quadrupole temperature 150℃; solvent delay 3.0 min; selected ion monitoring (SIM) mode. Optimized chromatographic and mass spectrometry parameters are shown in Table 2.

[0077] Table 2 Chromatographic and mass spectrometric analysis parameters for hazardous substances

[0078]

[0079] Note: * indicates quantitative ions.

[0080] 2.2 Results and Discussion

[0081] (1) Method Validation

[0082] Ultrasonic extraction is widely used due to its advantages such as high extraction efficiency, wide applicability, and simple operation. Referring to Li Hongyan's research on the extraction of volatile substances from mosquito repellent products, dichloromethane was used as the extraction solvent, and ultrasonic extraction for 30 minutes yielded the highest extraction efficiency. Therefore, this study adopted the above extraction conditions. The linear range, limit of determination, and repeatability of the method were verified. The results are shown in Table 3 below. The results in the table show that the method has a good linear range, correlation coefficient, and repeatability, and can be used to determine the content of high-risk substances in mosquito repellent patches.

[0083] Table 3. Method validation results

[0084]

[0085] (2) Measurement results

[0086] The established ultrasonic extraction combined with gas chromatography-mass spectrometry method was used to determine the content of selected positive samples. The results are shown in Table 4 below. The results show that the content of several substances in the positive samples is relatively high. If some of this content is released and inhaled by children, it could potentially cause harm. Therefore, research on their migration patterns is needed to further assess the risk level of their use.

[0087] Table 4 Results of high-risk substance detection in positive samples

[0088]

[0089] III. Research on the determination method of migration and release of volatile substances in mosquito repellent patches (method establishment)

[0090] 3.1 Experimental Section

[0091] (1) Instruments, reagents and materials

[0092] The instrument was a TSQ8000 Evo gas chromatograph-mass spectrometer (Thermo Fisher Scientific, USA), equipped with a thermal desorption unit (TDU) and a cold injection system (CIS) (Gerstel GmbH, Germany); an experimental chamber (4L volume, Tsinghua University); a flow meter (Defender 510L, BIOS, USA); and a sampling pump (Pocket Pump, SKC, USA).

[0093] α-pinene, D-limonene, eucalyptol, and other standards were purchased from Shanghai Anpu Experimental Technology Co., Ltd., with a purity ≥98%; methanol was chromatographically pure and purchased from Fisher Scientific, USA; Tenax TA desorption tubes were used (Gerstel, Germany).

[0094] (2) Preparation of standard solutions

[0095] Weigh 20 mg (accurate to 0.0001 g) of each standard into a 10 mL volumetric flask, dissolve and dilute to volume with methanol to prepare a single-standard stock solution with a mass concentration of 2000 mg / L, and store in a brown stock bottle (protected from light at 4°C). Prepare mixed stock solutions with a mass concentration of 100 mg / L using the single-standard stock solutions, and dilute with methanol to the appropriate concentration as needed before use.

[0096] (3) Sampling parameters for migration measurement

[0097] To more realistically simulate actual migration scenarios, volatile matter determination studies often employ experimental chambers, but commercially available experimental chambers are generally quite large (mostly 1m²). 3 (Sizes include 60L, etc.). Since mosquito repellent patches are small samples, large-volume experimental chambers are insufficient for accurately monitoring their volatile release process. Therefore, this study commissioned Tsinghua University to custom-make a 4L miniature experimental chamber, such as... Figure 4 As shown, this device is suitable for specialized research on the volatile release characteristics of samples such as mosquito repellent patches.

[0098] The experimental chamber includes a chamber body 1 and a cover 2 that covers it. A fan 3 is installed in the lower part of the chamber body 1, and the fan 3 is connected to an external power source via wires (which pass through the side wall of the chamber body 1). Multiple sample tray supports 4 (up to four) are fixed in the middle of the chamber body 1, on which grid-like sample trays 5 are placed. An air inlet pipe 6 is connected to the lower part of the chamber body 1, its lower end communicating with the internal space of the chamber body 1, and its upper end connected to an air source (providing air) via the air inlet pipe. An air outlet 7 is provided on the outside of the cover 2 (its lower end is connected to the internal space of the chamber body 1). The cover 2 has two sampling ports 8 (the lower end of which is connected to the internal space of the cabin 1), and an air outlet 7 (the upper end) is connected to a filter unit 9 (connected to the air inlet of the filter unit 9, the interior of the filter unit 9 is filled with filter material, such as graphite carbon, and the air outlet of the filter unit 9 is connected to the outside). A sealing cover is detachably provided on the sampling port 8 (the sealing cover is removed when sampling is needed, and the sealing cover is put on when sampling is not needed). A temperature sensor 10 (used to measure the internal temperature) is provided on the inside of the cover 2, and it is connected to an external power source through a wire (the wire passes through the cover 2).

[0099] The experimental chamber also includes sampling tubes (two pre-aged Tenax TA thermal desorption tubes). During sampling, the sealing cap is removed, and the sampling tubes are connected to sampling port 8 via a connector (the connector can be any commercially available product capable of connecting the two, such as silicone tubing). The chamber body 1 and the cover 2 are connected using adjustable sealing fasteners (common existing technology; tightening the cover 2 after it is closed makes the seal more secure, and loosening it when needed). A pressure valve and flow meter are installed on the air inlet pipe. The chamber body 1, cover 2, air inlet pipe, sealing cap, sample tray 5, and sample tray support 4 are all made of stainless steel.

[0100] The experimental chamber was placed in a pre-set temperature and humidity chamber (26℃, 50% humidity). The power to the experimental chamber fan 3 was turned on, and the fan began operating. The air circuit control switch was turned on, and the airflow rate was controlled at 500 mL / min via the pressure divider valve and flow meter. Air entered the experimental chamber at a flow rate of 500 mL / min through the inlet, and the fan 3 facilitated rapid diffusion within the chamber before exhausting through the outlet 7. After equilibration for 1 hour, the experimental chamber cover 2 was opened, and four mosquito repellent patches (selected positive samples) were placed on the sample tray 5. The cover 2 was then closed, and the sealing fasteners and the sealing cap were tightened. After the mosquito repellent patches were placed in the experimental chamber for equilibration for 0.5 hours, the volatile substances in the patches were collected after selecting different release times as needed. During collection, two aged Tenax tubes were used. The TA thermal desorption tubes were used as sampling tubes (aging conditions were referenced from the thermal desorption conditions). The inlet ends of the two thermal desorption tubes were connected to sampling port 8 via connectors, and the outlet ends were connected to the sampling pump via silicone tubes. The sampling pump was set to a sampling flow rate of 150 mL / min and a sampling time of 10 min. Under this program, 1500 mL of gas was collected from the experimental chamber. After sampling, the sampling tubes were removed. The thermal desorption tubes containing the collected samples were placed on the sample tray of the thermal desorption-gas chromatography-mass spectrometry (TCMS) instrument, and the experimental parameters were set for qualitative and quantitative analysis.

[0101] (4) Instruments and conditions

[0102] Thermal desorption conditions: sampling tube is Tenax TA, TDU temperature is 300℃, desorption time is 7min, cold trap CIS temperature is -10℃, and collection time is 8min;

[0103] Chromatographic conditions: HP-5MS column (30m×0.25mm×0.25μm); injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃ held for 1min, increased to 160℃ at 15℃ / min, then increased to 280℃ at 20℃ / min, held for 5min.

[0104] Mass spectrometry conditions: EI ionization mode, transfer line temperature 280℃; ion source temperature 230℃; quadrupole temperature 150℃; solvent delay 3.0 min; selected ion monitoring (SIM) mode. Optimized parameters are shown in Table 2.

[0105] 3.2 Results and Discussion

[0106] (1) Optimization of thermal desorption tube

[0107] Three representative desorption tubes commonly used for adsorbing volatile compounds were selected: Tenax TA, Tenax GR, and a combination tube (Tenax TA, Carbopack B, and Carbopack X). The adsorption effects of these three packing materials on α-pinene, D-limonene, and eucalyptol in mosquito repellent patches were investigated. First, one mosquito repellent patch was placed in a pre-aged 1L sampling bag, filled with 0.8L nitrogen gas, sealed, and left to stand at 26℃ for 4 hours. Two parallel samples were prepared for each sampling tube under the same conditions. Sampling was then performed at a flow rate of 100mL / min for 4 minutes. Thermal desorption analysis was performed immediately after sample collection. The results are shown below. Figure 2 As shown. From Figure 2 It can be seen that the three desorption tubes have strong adsorption capacity for α-pinene and D-limonene, while eucalyptol has relatively weak adsorption capacity due to its own properties. However, overall, Tenax TA has relatively good adsorption effect on each target analyte, so Tenax TA was selected for subsequent experimental optimization.

[0108] (2) Optimization of TDU and CIS conditions

[0109] The temperature and time of the thermal desorption unit (TDU) refer to the process of enriching the target analyte from the desorption tube to the cold trap at a certain temperature and time. The temperature and time of the cold trap CIS refer to the enrichment temperature maintained by the cold trap during TDU desorption and the time of release to the injection port. Therefore, the temperature and time of the TDU and the cold trap CIS determine the effectiveness of target analyte desorption in the desorption tube and cold trap enrichment. Thus, the desorption temperature and time of the TDU, and the enrichment temperature and time of the cold trap were optimized respectively. The results are as follows: Figure 3 As shown. First, the desorption time was set to 5 minutes, and the effect of different desorption temperatures (200, 220, 250, 270, 300℃) on the target analyte's response value was investigated. Figure 3 The TDU temperature unit showed that higher temperatures resulted in greater responses. Considering the sensitivity of the three substances, a TDU desorption temperature of 300℃ was selected. Then, with the desorption temperature set at 300℃, the effect of different desorption times (1, 3, 5, 7, 10 min) on the target analyte's response value was investigated. Figure 3As shown in the TDU time unit, the target analytes reached equilibrium after 7 minutes of desorption; therefore, the TDU desorption time was set to 7 minutes. Then, the TDU was set to desorption at 300℃ for 7 minutes, and the effects of CIS temperatures of -30, -20, -10, 0, and 10℃ on the target analyte response were investigated. Figure 3 The CIS temperature unit shows that the CIS temperature has little effect on the three substances, likely because these three substances are small-molecule and volatile, exhibiting good collection effects below 10℃. To make the cold trap collection more stable, the CIS temperature was set to -10℃. With the cold trap temperature set to CIS -10℃, the effect of different CIS collection times (3, 5, 8, 10, 15 min) on the target analyte response value was investigated. Figure 3 The CIS time unit shows that the target object has basically reached equilibrium after 8 minutes of supplementation, so the CIS supplementation time is set to 8 minutes.

[0110] (3) Optimization of sample sampling conditions

[0111] The number of mosquito repellent patches, sampling flow rate, and sampling time were investigated. More patches resulted in higher release at the same temperature; however, excessively high release levels could lead to breakthrough saturation in the desorption tube. Furthermore, the content determination results showed relatively high levels of α-pinene and D-limonene, which also resulted in relatively high release rates. Therefore, to avoid saturation in the desorption tube, four mosquito repellent patches were used. Sampling flow rate and sampling time are also important parameters affecting the method's sensitivity. Generally, slower flow rates and longer sampling times result in better adsorption in the desorption tube. However, to save time while ensuring adsorption efficiency, and referring to literature methods, the sampling flow rate was set to 150 mL / min, the sampling time to 10 min, and the sampling volume to 1500 mL.

[0112] (4) Method Validation

[0113] Under optimized conditions, the concentrations were measured sequentially from low to high. A standard curve was plotted with the peak area of ​​the quantitative ion as the ordinate and the mass concentration of the corresponding compound as the abscissa, and linear regression analysis was performed. The results are shown in Table 5 below. α-pinene and D-limonene showed good linearity in the range of 0.5–100 μg / L, with a correlation coefficient (r) of [missing value]. 2 The correlation coefficients (r) were all no less than 0.9988, and eucalyptol showed good linearity in the range of 1–100 μg / L, with correlation coefficients (r) of no less than 0.9988. 2 The values ​​were all 0.9981. The limits of detection (LODs) were calculated using a signal-to-noise ratio of 10, yielding LODs of 0.5, 0.5, and 1 ng for the three substances, respectively. Following the experimental conditions determined by this method, four mosquito repellent patches were placed in the experimental chamber for 2 hours before sampling. The experiment was repeated six times in parallel, and the precision of the method was calculated. The results are shown in Table 5. The method exhibits good repeatability and can be used for subsequent studies on migration patterns.

[0114] Table 5. Validation results of the migration method

[0115]

[0116] IV. A method for determining the content of volatile substances in mosquito repellent patches (actual sample testing method)

[0117] 4.1 Instruments, Reagents and Materials

[0118] 6890-5975 gas chromatograph-mass spectrometer (Agilent Technologies, USA); P300H ultrasonic cleaner (Elma GmbH, Germany); Nylon 66 filter membrane (0.22μm, Tianjin Jinteng Company).

[0119] 4.2 Sample Pretreatment

[0120] Take 0.1g of a mosquito repellent patch, cut it into 3mm×3mm fragments, put them into a 10mL small brown bottle, add 5mL of dichloromethane, seal tightly, and extract by sonication at room temperature for 30min. After sonication, place it at room temperature and then filter it through a membrane for analysis.

[0121] 4.3 Analysis Conditions

[0122] Chromatographic conditions: HP-5MS column, 30m×0.25mm×0.25μm; injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃, hold for 1 min, increase to 160℃ at 15℃ / min, then increase to 280℃ at 20℃ / min, hold for 5 min;

[0123] Mass spectrometry conditions: EI ionization mode, transfer line temperature 280℃; ion source temperature 230℃; quadrupole temperature 150℃; solvent delay 3.0 min; selected ion monitoring mode.

[0124] The volatile substances are α-pinene, D-limonene, and eucalyptol. The chromatographic and mass spectrometric analysis parameters are as follows:

[0125]

[0126] Note: * indicates quantitative ions.

[0127] V. A method for determining the actual release amount of volatile substances in mosquito repellent patches (actual sample testing method)

[0128] 5.1 Instruments, Reagents and Materials

[0129] The instrument included a TSQ8000 Evo gas chromatograph-mass spectrometer (Thermo Fisher Scientific, USA), equipped with a thermal desorption unit (TDU) and a cold injection system (CIS) (Gerstel GmbH, Germany); an experimental chamber (4L volume, Tsinghua University); a flow meter (Defender 510L, BIOS, USA); a sampling pump (Pocket Pump, SKC, USA); and Tenax TA desorption tubes (Gerstel GmbH, Germany).

[0130] Instruments such as Figure 4 As shown, the experimental chamber includes a chamber body 1 and a cover 2 that covers it. A fan 3 is installed in the lower part of the chamber body 1, and the fan 3 is connected to an external power source via a wire (the wire passes through the side wall of the chamber body 1). Multiple sample tray supports 4 (up to 4) are fixed in the middle of the chamber body 1, and grid-shaped sample trays 5 are placed on them. An air inlet pipe 6 is connected to the lower part of the chamber body 1, with its lower end connected to the internal space of the chamber body 1 and its upper end connected to an air source (providing air) via the air inlet pipe. An air outlet 7 is provided on the outside of the cover 2 (its lower end is connected to the internal space of the chamber body 1). The cover 2 has two sampling ports 8 (the lower end of which is connected to the internal space of the chamber 1), and an air outlet 7 (the upper end) is connected to a filter unit 9 (connected to the air inlet of the filter unit 9, the interior of the filter unit 9 is filled with filter material, such as activated carbon, and the air outlet of the filter unit 9 is connected to the outside). A sealing cover is detachably provided on the sampling port 8 (the sealing cover is removed when sampling is needed, and the sealing cover is put on when sampling is not needed). A temperature sensor 10 (used to measure the internal temperature) is provided on the inside of the cover 2, and it is connected to an external power source through a wire (the wire passes through the cover 2).

[0131] The experimental chamber also includes sampling tubes (two pre-aged Tenax TA thermal desorption tubes). During sampling, the sealing cap is removed, and the sampling tubes are connected to sampling port 8 via a connector (the connector can be any commercially available product capable of connecting the two, such as silicone tubing). The chamber body 1 and the cover 2 are connected using adjustable sealing fasteners (common existing technology; tightening the cover 2 after it is closed makes the seal more secure, and loosening it when needed). A pressure valve and flow meter are installed on the air inlet pipe. The chamber body 1, cover 2, air inlet pipe, sealing cap, sample tray 5, and sample tray support 4 are all made of stainless steel.

[0132] 5.2 Experimental Methods

[0133] Step (I): Place the experimental chamber in a pre-set temperature and humidity chamber (26℃, 50% humidity). Turn on the power to the experimental chamber fan 3. The fan 3 will start working. Turn on the air circuit control switch and control the air intake flow rate to 500 mL / min through the pressure divider valve and flow meter. Air will enter the experimental chamber at a flow rate of 500 mL / min from the air inlet. The fan 3 will cause the gas to diffuse rapidly within the experimental chamber, and then it will be discharged through the air outlet 7. After equilibration for 1 hour, open the experimental chamber cover 2, place 4 mosquito repellent patches on the sample tray 5, close the cover 2, and tighten the sealing fasteners and the sealing cap. After the mosquito repellent patches have been equilibrated in the experimental chamber for 0.5 hours, collect the volatile substances from the mosquito repellent patches after selecting different release times as needed. During collection, take two aged Tenax tubes. The TA thermal desorption tubes were used as sampling tubes (aging conditions were referenced from the thermal desorption conditions). The inlet ends of the two thermal desorption tubes were connected to sampling port 8 via connectors, and the outlet ends were connected to the sampling pump via silicone tubes. The sampling pump was set to a sampling flow rate of 150 mL / min and a sampling time of 10 min. Under this program, 1500 mL of gas was collected from the experimental chamber. After sampling, the sampling tubes were removed. The thermal desorption tubes containing the collected samples were placed on the sample tray of the thermal desorption-gas chromatography-mass spectrometry (TCMS) instrument, and the experimental parameters were set for qualitative and quantitative analysis.

[0134] Step (II): The migration and release of volatile substances in the mosquito repellent patch were determined by gas chromatography-mass spectrometry under the following analytical conditions:

[0135] Thermal desorption conditions: sampling tube is Tenax TA, TDU temperature is 300℃, desorption time is 7min, cold trap CIS temperature is -10℃, and collection time is 8min;

[0136] Chromatographic conditions: HP-5MS column, 30m×0.25mm×0.25μm; injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃, hold for 1min, increase to 160℃ at 15℃ / min, then increase to 280℃ at 20℃ / min, hold for 5min.

[0137] Mass spectrometry conditions: ionization mode was EI, transfer line temperature was 280℃; ion source temperature was 230℃; quadrupole temperature was 150℃; solvent delay was 3.0 min; and the monitoring mode was selected ion monitoring (SIM).

[0138] The volatile substances are α-pinene, D-limonene, and eucalyptol. The chromatographic and mass spectrometric analysis parameters are as follows:

[0139]

[0140] Note: * indicates quantitative ions.

[0141] VI. Results of Actual Sample Measurement

[0142] Using the established migration release assay method, five samples containing α-pinene, D-limonene, and eucalyptol, and exhibiting relatively high responses, were selected from 23 samples used in the initial positive sample screening. Their migration release was measured at real-time levels two hours after release, at a temperature of 26°C. The migration release results of the three volatile substances in the five samples are shown in Table 6.

[0143] Table 6 Results of migration and release measurement

[0144]

[0145] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for determining the content of volatile substances in mosquito repellent patches, characterized in that: The process includes the following steps: Sample pretreatment followed by analysis, under the following conditions: Chromatographic conditions: HP-5MS column, 30m×0.25mm×0.25μm; injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃, hold for 1 min, increase to 160℃ at 15℃ / min, then increase to 280℃ at 20℃ / min, hold for 5 min; Mass spectrometry conditions: EI ionization mode, transfer line temperature 280℃; ion source temperature 230℃; quadrupole temperature 150℃; solvent delay 3.0 min; selected ion monitoring mode.

2. The method for determining the content of volatile substances in mosquito repellent patches according to claim 1, characterized in that: The volatile substances are α-pinene, D-limonene, and eucalyptol, and the chromatographic and mass spectrometric analysis parameters are as follows: Note: * indicates quantitative ions.

3. The method for determining the content of volatile substances in mosquito repellent patches according to claim 2, characterized in that: The method also includes sample pretreatment: take 0.1g of a mosquito repellent patch, cut it into 3mm×3mm fragments, put it into a 10mL small brown bottle, add 5mL of dichloromethane, seal it tightly, and extract it by ultrasonication at room temperature for 30min. After ultrasonication, place it at room temperature and then filter it through a membrane for analysis.

4. A method for determining the actual release amount of volatile substances in a mosquito repellent patch, characterized in that: Includes the following steps: (I) Place the experimental chamber in a constant temperature chamber, and place the sample in the experimental chamber to simulate the natural environment. Take the sample after equilibration. (II) The migration and release of volatile substances in the mosquito repellent patch were determined by gas chromatography-mass spectrometry under the following analytical conditions: Thermal desorption conditions: sampling tube is Tenax TA, TDU temperature is 300℃, desorption time is 7min, cold trap CIS temperature is -10℃, and collection time is 8min; Chromatographic conditions: HP-5MS column, 30m×0.25mm×0.25μm; injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃, hold for 1 min, increase to 160℃ at 15℃ / min, then increase to 280℃ at 20℃ / min, hold for 5 min; Mass spectrometry conditions: ionization mode was EI, transfer line temperature was 280℃; ion source temperature was 230℃; quadrupole temperature was 150℃; solvent delay was 3.0 min; and the monitoring mode was selected ion monitoring (SIM).

5. The method for determining the actual release amount of volatile substances in mosquito repellent patches according to claim 4, characterized in that: The volatile substances are α-pinene, D-limonene, and eucalyptol, and the chromatographic and mass spectrometric analysis parameters are as follows: Note: * indicates quantitative ions.

6. The method for determining the actual release amount of volatile substances in mosquito repellent patches according to claim 5, characterized in that: The experimental chamber includes a chamber body (1) and a cover (2) covering the upper part of the chamber body (1). A fan (3) is installed in the lower part of the chamber body (1), and the fan (3) is connected to an external power source through a wire. Multiple sample tray supports (4) are fixed in the middle of the chamber body (1), and a grid-shaped sample tray (5) is placed on them. An air inlet pipe (6) is connected to the lower part of the chamber body (1), with its lower end connected to the internal space of the chamber body (1) and its upper end connected to an air source through the air inlet pipe. An air outlet (7) and two sampling ports (8) are provided on the outside of the cover (2). The air outlet (7) is connected to a filter unit (9). A sealing cover is detachably provided on the sampling port (8). A temperature sensor (10) is provided on the inside of the cover (2), which is connected to an external power source through a wire.

7. The method for determining the actual release amount of volatile substances in mosquito repellent patches according to claim 6, characterized in that: The experimental chamber also includes a sampling tube. During the sampling process, the sealing cover is removed and the sampling tube is connected to the sampling port (8) through a connector. The chamber body (1) and the cover body (2) are connected by an adjustable sealing fastener. A pressure divider valve and a flow meter are provided on the air inlet pipe.

8. The method for determining the actual release amount of volatile substances in mosquito repellent patches according to claim 7, characterized in that: Step (I) specifically includes the following steps: The experimental chamber was placed in a constant temperature and humidity chamber with a set temperature and humidity of 26°C and 50%. The power supply of the experimental chamber fan (3) was turned on, and the fan (3) started working. The air circuit control switch was turned on, and the air intake flow rate was controlled to 500 mL / min through the pressure divider valve and flow meter. The air entered the experimental chamber from the air inlet at a flow rate of 500 mL / min. The fan (3) made the gas diffuse rapidly in the experimental chamber and then discharged it through the air outlet (7). After balancing in this state for 1 hour, the experimental chamber cover (2) was opened, and 4 mosquito repellent patches were placed on the sample tray (5). The cover (2) was closed, and the sealing fasteners and the sealing cap were tightened. After the mosquito repellent patches were placed in the experimental chamber for balancing for 0.5 hours, the volatile substances in the mosquito repellent patches were collected after selecting different release times according to the requirements. When collecting, two aged Tenax tubes were taken. The TA thermal desorption tube is used as the sampling tube. The inlet end of the two thermal desorption tubes is connected to the sampling port (8) through the connector, and the outlet end is connected to the sampling pump through the silicone tube. The sampling flow rate is set to 150 mL / min and the sampling time is 10 min. Under this program, 1500 mL of gas in the experimental chamber is collected. After sampling, the sampling tube is removed. The thermal desorption tube with the collected sample is placed on the sample tray of the thermal desorption-gas chromatography-mass spectrometry instrument, and the experimental parameters are set for qualitative and quantitative analysis.

9. A method for screening positive samples from mosquito repellent patches, characterized in that: Includes the following steps: (A) Sample pretreatment: Crush the sample into particles smaller than 5 mm, weigh 0.1 g into a 20 mL headspace vial, seal it, and place it into a headspace sampler; (B) Analysis conditions: Chromatographic conditions: HP-5MS column, 30m×0.25mm×0.25μm; injection port temperature 250℃; carrier gas high-purity helium, flow rate 1mL / min; split injection, split ratio 20:1; column oven temperature program: initial temperature 40℃, hold for 1 min, increase to 160℃ at 15℃ / min, then increase to 280℃ at 20℃ / min, hold for 5 min; Mass spectrometry conditions: ionization mode: EI; transfer line temperature: 280℃; ion source temperature: 230℃; quadrupole temperature: 150℃; monitoring mode: full scan monitoring; scan range: 40-500 m / z. Headspace conditions: equilibrium temperature 60℃, equilibrium time 30 min, quantitative loop temperature 100℃, transfer line temperature 120℃, quantitative loop volume 1 mL, injection time 30 s; (C) Screening positive samples: After obtaining the analysis results, the samples containing α-pinene, D-limonene and eucalyptol were sorted by peak area, and the samples in which the peak areas of all three substances were above the median value were selected as positive samples.

10. A method for determining the content of volatile substances in mosquito repellent patches according to any one of claims 1 to 3, or a method for establishing a method for determining the actual release amount of volatile substances in mosquito repellent patches according to any one of claims 4 to 8, characterized in that: Includes the following steps: (a) Preparation of standard solutions; (b) Screening positive samples of mosquito repellent patches, specifically as described in claim 9; (c) Optimize various experimental parameters.

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