Detection method of N-(2-methylcyclohexyl)-2, 3, 4, 5, 6-pentafluoro-benzamide and application of N-(2-methylcyclohexyl)-2, 3, 4, 5, 6-pentafluoro-benzamide

Abstract

The invention discloses a method for detecting N-(2-methylcyclohexyl)-2, 3, 4, 5, 6-pentafluoro-benzamide and application of the method, and belongs to the technical field of detection.The method comprises the steps that a series of standard solutions are prepared, the concentrations of target objects in different standard solutions are different, the series of standard solutions are detected through an ultra-high performance liquid chromatography, and the content of the target objects in the standard solutions is determined according to the content of the N-(2-methylcyclohexyl)-2, 3, 4, 5, 6-pentafluoro-benzamide. A standard curve is obtained according to a detection result, a to-be-detected sample is pretreated to obtain a to-be-detected solution, the to-be-detected sample is detected by adopting ultra-high performance liquid chromatography, and the target object content of the to-be-detected sample is calculated according to a linear equation of the standard curve. The method can effectively detect the content of N-(2-methylcyclohexyl)-2, 3, 4, 5, 6-pentafluoro-benzamide, has the advantages of high precision, good accuracy, high sensitivity, strong anti-interference capability and the like, and can be used for quality control of products containing N-(2-methylcyclohexyl)-2, 3, 4, 5, 6-pentafluoro-benzamide.

Description

Technical Field

[0001] The invention belongs to the technical field of sweetener detection, and relates to a detection method and application of N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide. Background Art

[0002] N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide (CAS#1003050-32-5) is a synthetic fragrance substance with the following structural formula.

[0003]

[0004] It has been approved as "Generally Recognized as Safe" (GRAS) by the Flavor and Natural Extracts Manufacturers Association (FEMA), FEMA No. 4678. JECFA (Joint Expert Group on Food Additives of WHO and FAO) evaluated the substance (JECFA No. 2081) and believed that the substance has no safety issues in the current intake level, in various foods and e-cigarettes, as a flavoring, and can be used in appropriate amounts according to production needs. The European Food Safety Authority (ESFA) evaluated the safety of the substance and registered it as FL16.119.

[0005] In developed countries, the increasing consumption of sugar is closely related to obesity and early type 2 diabetes. Therefore, the synthetic sweetener N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide (hereinafter referred to as sweetener No. 4678) can be used as a potential sweetness regulator in food, medicine and other consumer products that require sweetness regulation.

[0006] Electronic atomization liquid is the core component of electronic cigarettes. It forms aerosols to deliver nicotine and other ingredients to the respiratory system. Sweeteners are an important component of electronic atomization liquid, which can optimize the taste and enhance the consumer experience. Currently, the commonly used sweetener for electronic atomization liquid is neotame. Preliminary studies have shown that the atomization rate and stability of neotame are lower than expected, while the atomization rate of sweetener No. 4678 can be as high as 99%. Therefore, sweetener No. 4678 can be used as a potential alternative sweetener for electronic atomization liquid. In order to further study the applicability of this sweetener in oral consumer products, the first issue to be solved is whether the content of sweetener No. 4678 in oral consumer products can be effectively detected.

[0007] However, there is currently no reference method to detect the content of sweetener No. 4678 in oral consumer products such as electronic atomizer liquid, food, and oral products. Summary of the invention

[0008] The invention aims to provide a method for detecting N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide and application thereof. The method can effectively detect the content of N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide and has high precision and good accuracy, and can be used for quality control of products containing N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present application discloses a method for detecting N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide, comprising: preparing a series of standard solutions, wherein different standard solutions contain different concentrations of a target substance, wherein the target substance is N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide; detecting a series of the standard solutions by ultra-high performance liquid chromatography, and obtaining a standard curve according to the detection results; pretreating a sample to be tested to obtain a solution to be tested; detecting the solution to be tested by ultra-high performance liquid chromatography, and determining the concentration of N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide in the sample to be tested according to the detection results and the standard curve; the conditions of the ultra-high performance liquid chromatography include: a detection wavelength of 210-220 nm.

[0011] In one implementation of the present application, the pretreatment of the sample to be tested to obtain the solution to be tested includes: when the sample to be tested is liquid, obtaining a uniformly mixed sample to be tested, and placing the sample to be tested in a centrifuge tube; adding an extraction solvent to the centrifuge tube to make up the volume, then performing oscillation extraction for 20 to 30 minutes, taking the supernatant, and filtering to obtain the solution to be tested; when the sample to be tested is solid, obtaining the sample to be tested, crushing the sample to be tested, and placing the crushed sample to be tested in a centrifuge tube, adding an extraction solvent to the centrifuge tube to make up the volume, then performing oscillation extraction for 20 to 30 minutes, taking the supernatant, and filtering to obtain the solution to be tested.

[0012] In one implementation of the present application, the extraction solvent is an organic solvent, preferably a pure solvent of ethanol, acetonitrile and methanol, or a mixed solution of the organic solvent and water.

[0013] In one implementation of the present application, the chromatographic column used in the ultra-high performance liquid chromatography is an octadecylsilane bonded silica gel chromatographic column or a chromatographic column equivalent to the octadecylsilane bonded silica gel chromatographic column; preferably, the chromatographic column is an end-capped type.

[0014] In one implementation of the present application, the conditions of the ultra-high performance liquid chromatography include: column temperature 20-35°C; flow rate 0.3mL / min to 1mL / min; injection volume 2-10μL.

[0015] In one implementation of the present application, the conditions of the ultra-high performance liquid chromatography include that the elution program is gradient elution, and the gradient is as follows: 0-10 min, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%; 10 min, the volume fraction of mobile phase A is 5%, and the volume fraction of mobile phase B is 95%; 13 min, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%; wherein, mobile phase A is ultrapure water and mobile phase B is acetonitrile.

[0016] In one implementation of the present application, the conditions of the ultra-high performance liquid chromatography method include: the chromatographic column is a Poroshell 120EC-C18 chromatographic column or a chromatographic column equivalent to Poroshell 120EC-C18.

[0017] In one implementation of the present application, the conditions of the ultra-high performance liquid chromatography include: the chromatographic column is Agilent5HC-C18 chromatographic column or its equivalent chromatographic column, the elution procedure isocratic elution, mobile phase A is ultrapure water, and mobile phase B is acetonitrile: mobile phase A: mobile phase B = 3~4.5:5.5~7.

[0018] The second aspect of the present application provides the use of the above detection method in the quality control of products containing N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide; the products include oral consumer products.

[0019] Beneficial Effects of the Invention

[0020] (1) The detection method provided by the present invention can effectively detect the content of N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide with high precision and good accuracy, and can be used for quality control of oral consumer products containing N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide.

[0021] (2) The sample pretreatment process of the present invention is simple and convenient. The UPLC detection method can accurately determine the content of N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide in various foods, and the detection time is short, which can achieve rapid determination and make up for the lack of N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide detection method in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This is the standard curve diagram of sweetener No. 4678 involved in this application;

[0023] Figure 2 It is a chromatogram of the quality control sample for testing sweetener No. 4678 involved in this application;

[0024] Figure 3 The sample superimposed comparison chromatogram of the electronic atomized liquid and QC solution for detecting apple and pear flavors involved in this application;

[0025] Figure 4 The chromatogram of the electronic atomizing liquid for detecting menthol flavor involved in the present application;

[0026] Figure 5 This is a chromatogram overlay comparison diagram of sweetener No. 4678 in the detection of food and quality control samples involved in this application;

[0027] Figure 6 This is a chromatogram overlay comparison diagram of the sample of Example 4 involved in the detection of the present application;

[0028] Figure 7 This application relates to a chromatogram of the sample of comparative example 1;

[0029] Figure 8 This application relates to the chromatogram of the sample of comparative example 2;

[0030] Fig. 9 This application relates to the chromatogram of the sample of comparative example 3;

[0031] Fig.10 This is a chromatogram overlay comparison diagram of the sample and the quality control sample of the detection comparative example 4 involved in the present application;

[0032] Fig.11 This is a chromatogram overlay comparison diagram of the quality control sample of the sample of comparative example 5 involved in the detection of the present application;

[0033] Fig.12 This is a chromatogram overlay comparison diagram of the quality control sample of the sample of comparative example 6 involved in the detection of the present application;

[0034] Fig.13 This is a chromatogram overlay comparison diagram of the quality control sample of the sample of comparative example 7 involved in the detection of the present application;

[0035] Fig.14 This application relates to the absorption curve diagram of sweetener No. 4678 in the detector. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can effortlessly recognize that some of the features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations according to the description in the specification and the general technical knowledge in the art.

[0037] It should be noted that the oral consumables in this application include but are not limited to foods, beverages, electronic atomizer liquids, oral care products, and medicines that need to be sweetened and enter or potentially enter the oral cavity. The state of oral consumables includes solid, liquid, semi-solid, etc. These products usually have complex ingredients, so it is necessary to develop a detection method with strong anti-interference.

[0038] The present application relates to a detection method.

[0039] In a specific embodiment, the target substance to be detected may be a sweetener in an orally consumable product.

[0040] In a specific embodiment, the target object to be detected may be a sweetener in a novel tobacco product.

[0041] In a specific embodiment, the target object to be detected may be a sweetener in the electronic atomization liquid.

[0042] In a specific embodiment, the target object to be detected may be a sweetener in biscuits.

[0043] In a specific embodiment, the target object to be detected may be a sweetener in hard candy.

[0044] In a specific embodiment, the target object to be detected may be a sweetener in a beverage.

[0045] In one specific embodiment, the target substance detected is sweetener No. 4678.

[0046] In a specific embodiment, the detection method may refer to a method for detecting sweeteners in orally consumable products.

[0047] In a specific embodiment, the detection method may refer to the detection method of sweetener No. 4678 in oral consumables. It should be noted that oral consumables may be solid foods, such as candy, biscuits, bread, etc.; liquid foods, such as beverages, juices, dairy products, etc.; oral products, such as mouthwash, toothpaste, dental floss containing detergents, etc.; new tobacco products, such as electronic cigarette atomizers, snus, smoking cessation candies, etc.; and other consumer products that require sweetness adjustment and are used or eaten in the mouth.

[0048] In a specific embodiment, the electronic atomization liquid refers to a liquid matrix used in an electronic atomizer to deliver nicotine or other active ingredients to the respiratory system. It should be noted that the electronic atomization liquid may include propylene glycol, glycerin, nicotine or nicotine salts or nicotine derivatives, flavors and sweeteners, etc.

[0049] In a specific embodiment, the electronic atomization liquid can be an electronic atomization liquid of various flavors. For example, it can be tobacco flavor, fruit flavor, herbal flavor, beverage flavor, etc. Among them, fruit flavors include apple, banana, blueberry, strawberry, watermelon and other flavors. Herbal flavors include mint, fennel, holly, vanilla, cinnamon and other flavors. Beverage flavors include cola, Red Bull and other flavors.

[0050] In a specific embodiment, in preparing a series of standard solutions, the concentration of the target substance (sweetener No. 4678) in different standard solutions is different. The "concentration" in this application can be a mass volume ratio of mg / L. It should be noted that when preparing the standard solution, the present application found that sweetener No. 4678 is insoluble in water, but has good solubility in organic solvents including ethanol, methanol, and acetonitrile systems; it can also be dissolved in a mixed solution system of organic solvents and water.

[0051] In a specific embodiment, in preparing a series of standard solutions, sweetener No. 4678 can be first dissolved in ethanol to form a high concentration (referring to a concentration of about 1000 mg / L), and then diluted with ethanol to an intermediate concentration (100 mg / L), and then a standard curve is established.

[0052] In a specific embodiment, the concentration of the target substance (Sweetener No. 4678) in a series of standard solutions can be from 0 mg / L to 100 mg / L. It should be noted that, thus, the standard curve can cover the concentration range of Sweetener No. 4678 that may exist in oral consumable products.

[0053] In a specific embodiment, the concentrations of the target substance (sweetener No. 4678) in a series of standard solutions are 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, and 100 mg / L, respectively.

[0054] In a specific embodiment, the sample pretreatment of the electronic atomization liquid to be tested includes: mixing the electronic atomization liquid with ethanol. It should be noted that the main components of the electronic atomization liquid are propylene glycol and glycerol, etc., which need to be dissolved using a polar solvent; in this application, ethanol is used to extract the electronic atomization liquid, which has better environmental protection characteristics than organic solvents such as methanol and acetonitrile.

[0055] In a specific embodiment, the pretreatment of the electronic atomized liquid sample to be tested further includes: obtaining uniform electronic atomized liquid to be tested, placing the electronic atomized liquid to be tested in a centrifuge tube, and constant volume with ethanol to obtain a solution to be tested.

[0056] In a specific embodiment, the pretreatment of the electronic atomized liquid sample to be tested further includes: after the volume is fixed with ethanol, the centrifuge tube is vortexed and mixed at a speed of 2500-2900 rpm / min for 10-30 minutes. It should be noted that this operation can fully extract the electronic atomized liquid to be tested.

[0057] In a specific embodiment, the pretreatment of the electronic atomized liquid sample to be tested also includes filtering the solution to be tested using a 0.45 μm organic filter membrane. It should be noted that filtering the sample using a 0.45 μm organic filter membrane can remove tiny particles, dust and insoluble impurities in the sample, which is beneficial to protect instrument components such as chromatographic columns and detectors on the one hand; on the other hand, it can also improve the stability and reproducibility of the detection, reduce ghost peaks and noise caused by particulate matter, and improve data quality.

[0058] In a specific embodiment, the candy sample to be tested is pretreated, including crushing the candy sample, then obtaining the crushed sample and placing it in a centrifuge tube, adding ethanol to the fixed volume, placing it in a vortex oscillator under sealed conditions, and oscillating and extracting at 2500-2900 rpm / min for 20-30 minutes. It should be noted that this operation can fully extract the sweetener No. 4678 in the candy to be tested.

[0059] In a specific embodiment, the pretreatment of the biscuit sample to be tested includes crushing the biscuit sample, then obtaining the crushed sample and placing it in a centrifuge tube, adding ethanol to the fixed volume. Under sealed conditions, the sample is placed in a vortex oscillator and oscillated at 2500 to 2900 rpm / min for 20 to 30 minutes. It should be noted that this operation can fully extract the sweetener No. 4678 in the biscuit to be tested.

[0060] In a specific embodiment, the pretreatment of the beverage sample to be tested includes placing the beverage sample in a centrifuge tube, adding ethanol to a fixed volume, placing the beverage sample in a vortex shaker under a sealed condition, and oscillating and extracting at 2500-2900 rpm / min for 20-30 minutes. It should be noted that this operation can fully extract the sweetener No. 4678 in the beverage to be tested.

[0061] In a specific embodiment, ultra-high performance liquid chromatography can be used to detect the test solution and the standard solution.

[0062] In a specific embodiment, an ultra-high performance liquid chromatograph can be used to detect the test solution and the standard solution.

[0063] In a specific embodiment, an ultra-high performance liquid chromatograph may be used to detect the standard solution to obtain a standard curve.

[0064] In a specific embodiment, an ultra-high performance liquid chromatograph can be used to detect the test solution, and the concentration of the target substance in the test solution can be obtained based on the test result and the standard curve, thereby obtaining the concentration or content of the target substance in the test sample.

[0065] In a specific embodiment, the chromatographic column used in the ultra-high performance liquid chromatography method is an octadecylsilane bonded silica gel chromatographic column or a chromatographic column equivalent to the octadecylsilane bonded silica gel chromatographic column.

[0066] In a specific embodiment, the chromatographic column used in the ultra-high performance liquid chromatography method is an end-capped chromatographic column.

[0067] In a specific embodiment, the chromatographic column used in the ultra-high performance liquid chromatography method is a Poroshell 120EC-C18 chromatographic column or a chromatographic column equivalent to Poroshell 120EC-C18.

[0068] In a specific embodiment, the chromatographic conditions in the ultra-high performance liquid chromatography method are as follows: the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is ultrapure water, and mobile phase B is acetonitrile.

[0069] In a specific embodiment, the chromatographic conditions in the ultra-high performance liquid chromatography method are: the elution program used is gradient elution. It should be noted that the use of gradient elution can shorten the analysis cycle, improve the separation ability, improve the peak shape, reduce tailing and increase the detection sensitivity.

[0070] In a specific embodiment, the chromatographic conditions in the ultra-high performance liquid chromatography are as follows: the gradient elution process is as follows: the initial mobile phase is 95% ultrapure water and 5% acetonitrile, which is maintained for 10 minutes; then the acetonitrile concentration is increased to 95%, and the ultrapure water concentration is reduced to 5%, which is maintained for 2 minutes; then the acetonitrile concentration is reduced to 5%, and the ultrapure water concentration is increased to 95%, which is maintained for 1 minute, and the elution procedure is terminated.

[0071] In a specific embodiment, the conditions of ultra-high performance liquid chromatography are: the chromatographic column Agilent5HC-C18 chromatographic column is or an equivalent chromatographic column, the elution procedure isocratic elution, the mobile phase A is ultrapure water, and the mobile phase B is acetonitrile: mobile phase A: mobile phase B = 3 to 4.5: 5.5 to 7. It should be noted that under this condition, the separation effect of the target in the medium with relatively simple components is good, and it can be separated in a shorter time.

[0072] In a specific embodiment, the chromatographic conditions in the ultra-high performance liquid chromatography method are: the injection volume is 2 μL to 10 μL. Preferably, the injection volume is 5 μL.

[0073] In a specific embodiment, the chromatographic conditions in the ultra-high performance liquid chromatography method are: the column temperature is 25° C. to 30° C. Preferably, the column temperature is 30° C.

[0074] In a specific embodiment, the chromatographic conditions in the ultra-high performance liquid chromatography method are: a flow rate of 0.3 mL / min to 1 mL / min. Preferably, the flow rate is 0.3 mL / min.

[0075] In a specific embodiment, the chromatographic conditions in the ultra-high performance liquid chromatography method are: the detection wavelength is 200nm to 220nm. Preferably, the detection wavelength is 210-220nm; more preferably, the detection wavelength is 218nm; Fig.14 This application relates to the ultraviolet absorption scanning diagram of sweetener No. 4678. It should be noted that when the applicant explored the ultraviolet absorption wavelength of sweetener No. 4678, Fig.14 As shown, it can be found that the target has no clear characteristic absorption. After further exploration, it is found that the absorption is better in the range of 200-220nm. However, considering the absorption of the solvent, the final screening of the wavelength in the range of 210-220nm can achieve the anti-interference effect, among which the effect at 218nm is the best.

[0076] In a specific embodiment, in the detection method of the present application, a test result of the peak time (retention time) of sweetener No. 4678 is 10.349 min.

[0077] In a specific embodiment, in the detection method of the present application, sweetener No. 4678 has two absorption peaks.

[0078] In a specific embodiment, the detection method of the present application can detect a minimum content of 3.5 μg / g of sweetener No. 4678 in oral consumable products.

[0079] In a specific embodiment, in the detection method of the present application, the lowest content of sweetener No. 4678 in the orally consumed product that can be quantified is 11.8 μg / g.

[0080] The present application is further described in detail below by specific experimental process and experimental data examples. The following examples only further illustrate the present application and should not be construed as limiting the present application. In the present embodiment, unless otherwise specified, the reagents and instruments used are all commercially available, and the experimental operation is carried out in accordance with the product specification and conventional experimental specifications.

[0081] Example 1

[0082] The instrument parameters used in this example are shown in Table 1 below:

[0083] Table 1

[0084]

[0085]

[0086] Establishing a standard curve: Prepare a series of standard solutions with the target substance (sweetener No. 4678) concentrations of 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, and 100 mg / L, and perform ultra-high performance liquid chromatography on each standard solution according to the parameters in Table 1 above to obtain the peak areas of the target substance. The results are shown in Table 2 below:

[0087] Table 2

[0088]

[0089] A standard curve was established with the concentration of the target substance (sweetener No. 4678) as the horizontal axis and the peak area as the vertical axis. Figure 1 This application relates to the standard curve of sweetener No. 4678. The linear equation of the standard curve of sweetener No. 4678 is Y=21.71538X+14.87546, R 2 is 0.99992>0.999. It can be seen that the target transfer curve has good linearity and R 2 Satisfies the requirement of >0.999.

[0090] Limit of detection and limit of quantitation: The limit of detection (LOD) refers to the concentration of the substance being measured corresponding to the detection limit of the method when the detection method is operated. The limit of quantitation (LOQ) refers to the concentration of the substance being measured corresponding to the lower limit of quantitation when the detection method is operated. In the method of the present application, a low-concentration mixed standard working solution is injected into an ultra-high performance liquid chromatography, and the limit of detection LOD is calculated at a 3-fold signal-to-noise ratio (S / N=3), and the limit of detection LOQ is calculated at a 10-fold signal-to-noise ratio (S / N=10). The results are shown in Table 3 below.

[0091] Table 3

[0092] Target substance Original value (mg / L) LOD theoretical value (μg / g) LOQ theoretical value (μg / g) Sweetener No. 4678 5.05 3.5 11.8

[0093] Quality control sample testing: ensure the accuracy and repeatability of the experiment and the accuracy and stability of the instrument. Quality control sample: a high-concentration QC solution (containing sweetener No. 4678) of the standard curve prepared with the mother solution homologous to the standard curve. Figure 2 This is a chromatogram of the quality control sample of the sweetener No. 4678 involved in the present application. It should be noted that in the chromatogram, the target sweetener No. 4678 has two characteristic peaks, which may be isomers after analysis. Figure 2 As shown, the peak time of the target sweetener No. 4678 is 10.349 min, and the peak shape is good. Among them, the one with the largest peak area is the QC solution (content is 100 mg / L).

[0094] Sample treatment: Weigh 0.2 g of the sample to be tested and place it in a 15 mL centrifuge tube, add 10 mL of 95% ethanol solution, vortex and oscillate for 20 min to extract, take 1 mL of the extract and filter it through a 0.45 μm organic filter membrane and place it in an injection vial for ultra-high performance liquid chromatography detection. The ultra-high performance chromatography is performed according to the parameters in Table 1.

[0095] Calculation formula for the content of the target substance in the sample to be tested

[0096] According to the ultra-high performance liquid chromatography detection method, the content of sweetener No. 4678 in the sample is calculated according to formula 1:

[0097]

[0098] in,

[0099] C x represents sample concentration, mg / g;

[0100] C r Represents the concentration of the sample to be tested, mg / L;

[0101] C0 represents the concentration of blank sample, mg / L;

[0102] m represents the weight of the sample to be tested, g.

[0103] Precision verification: Prepare standard samples of known concentration according to the sample processing method, and measure the standard samples of known concentration 6 times to determine their precision. The results are shown in Table 4 below

[0104] Table 4

[0105]

[0106] As can be seen from Table 4, the precision of sweetener No. 4678 is 0.63%, which meets the requirement of less than <10%, indicating that the precision of this method is good.

[0107] Accuracy verification: The accuracy of the method was examined by spike recovery. Take a sample S1 (the solvent is a PGVG mixed solution) and add the three concentration levels of the standard curve at low, medium and high levels, three parallels at each concentration level, and a single injection. The recovery rate was calculated according to Formula 2.

[0108] Verification standard: The recovery rate data should be between 90.0 and 110.0%.

[0109]

[0110] C test : Content of sample after spike (mg / kg);

[0111] C0: content of sample before spike (mg / kg);

[0112] C spiked : Added amount (mg).

[0113] The results are shown in Table 5 below.

[0114] Table 5

[0115]

[0116]

[0117] As can be seen from Table 5, the recovery rate of sweetener No. 4678 is between 95.8% and 98.7%, meeting the requirement of 90% to 110%. This indicates that the detection method of the present application has good accuracy and can be used for quantitative detection of sweetener No. 4678 in oral consumables.

[0118] The chromatogram of sample S1 is: Figure 2 The smaller the peak area, the Figure 2It can be seen that the peak time of the S1 sample is consistent with the peak time of the QC solution. It should be noted that the S1 sample here is a self-prepared simple atomization sample containing sweetener No. 4678, that is, a mixed solution of PG (propylene glycol): VG (vegetable glycerin) = 1:1 is used as the solvent, and sweetener No. 4678 is dissolved in PGVG solution to prepare a sample that can be atomized; wherein PGVG solution is the main solvent of the electronic atomization liquid. Figure 2 It can be seen that the solvent PGVG solution did not interfere with the target sweetener No. 4678, indicating that the method has good selectivity.

[0119] Example 2 Actual sample detection

[0120] Two flavors of electronic atomizing liquid were prepared, namely apple pear flavor and menthol flavor; the electronic atomizing liquid contained PGVG solution, sweetener No. 4678, flavor and nicotine. The electronic atomizing liquid of the two flavors was processed according to the "sample processing to be tested" steps and the ultra-high performance liquid chromatography determination was completed according to the parameters in Table 1.

[0121] Figure 3 This is a sample superimposed comparison chromatogram of the electronic atomized liquid and QC solution for detecting apple and pear flavors involved in this application. Figure 3 It can be seen that in a complex system, the target substance (sweetener No. 4678) is less interfered by miscellaneous peaks and has a good peak shape, which is conducive to quantification. Therefore, the apple-pear flavored electronic atomization liquid is tested using this method, which has strong anti-interference ability and good selectivity.

[0122] Figure 4 This is a chromatogram of the electronic atomization liquid for detecting menthol flavor involved in the present application, from Figure 4 It can be seen that the peak time of the target substance (sweetener No. 4678) is 10.356min, with two characteristic peaks, which is basically consistent with the peak time of the quality control sample, with good peak shape, which is conducive to quantification. Therefore, the menthol-flavored electronic atomization liquid is tested using this method, with strong anti-interference ability and good selectivity.

[0123] Example 3

[0124] Three different food samples of a certain brand on the market were randomly selected, namely hard candy, biscuits and beverages, and the application method of this application was used for application testing.

[0125] Among them, the processing method of solid samples is as follows:

[0126] The solid sample was crushed, and then 0.2 g of the crushed sample (accurate to 0.0001 g) was weighed and placed in a 15 mL centrifuge tube. 10 mL of ethanol solution was added, and the tube was sealed and placed in a vortex oscillator for 20 min of oscillation extraction. After standing, the supernatant was filtered through a 0.45 μm organic phase filter membrane for testing and transferred to a 2 mL brown sample bottle for standby use. Then, the ultra-high performance liquid chromatography determination was completed according to the parameters in Table 1.

[0127] The liquid sample was processed according to the "sample processing" step in Example 1 and the ultra-high performance liquid chromatography determination was completed according to the parameters in Table 1.

[0128] The test results are shown in Table 6 and Figure 5 shown.

[0129] Table 6

[0130] Sample name biscuit Hard Candy drinks Detection content (mg / L) ND ND ND

[0131] ND in Table 6 means not detected. It should be noted that sweetener No. 4678 is a new synthetic sweetener, which has been proven to be safe for use in food additives and is a potential sweetener substitute. However, it has not been officially approved for use in my country, so it is reasonable that the target substance was not detected in the sample. Figure 5 This is a superimposed comparison diagram of sweetener No. 4678 in the food tested in this application and the quality control sample. Figure 5 It can be seen that none of the three food samples obtained the corresponding characteristic peaks of sweetener No. 4678, which means that sweetener No. 4678 was not added to the three food samples tested.

[0132] Example 4

[0133] A peach-flavored electronic atomization liquid was prepared and the target (sweetener No. 4678) was detected using the method of the present application. The difference is that ethanol and acetonitrile were used as extraction solvents in the sample pretreatment, and the pretreatment extraction effects of different extraction solvents on the target were analyzed. The results are shown in Table 7 and Figure 6 shown.

[0134] Table 7

[0135] Extraction reagents Acetonitrile Ethanol RSD / % Detection content (mg / L) 22.41 22.65 0.75

[0136] From Table 7, it can be seen that the content of the target substance (sweetener No. 4678) in the electronic atomization liquid with apple and pear flavor under two different extraction solvents calculated by the linear equation of the standard curve and Formula 1, wherein the relative standard deviation is 0.75%, indicating that the two extraction solvents have good extraction consistency for the target substance.

[0137] The present application relates to the detection of superimposed chromatograms of sweetener No. 4678 in the sample of Example 4 under different extraction solvents. Figure 6 It can be seen that the peak time and peak area of ​​the target substance (sweetener No. 4678) corresponding to the samples treated with the two extraction solvents are basically the same.

[0138] Comparative Example 1

[0139] The difference between Comparative Example 1 and Example 1 is that the initial mobile phase ratio in the detection parameters of ultra-high performance liquid chromatography is 50% water and 50% acetonitrile. The effect of different mobile phase ratios on the separation of the target object was tested to screen the optimal detection parameters. Figure 7 As shown, from Figure 7 It can be seen that the characteristic peaks of the target substance and other substances appear at the same time and cannot be separated.

[0140] Comparative Example 2

[0141] The difference between Comparative Example 2 and Example 1 is that the chromatographic column in the ultra-high performance liquid chromatography detection parameters is Agilent 5HC-C18 (2) (4.6 × 250 mm, 5 μm) column for detection, the flow rate is 1 mL / min; the isocratic elution method is adopted, and the mobile phase ratio is 10% water and 90% acetonitrile. By testing the effects of different lengths of chromatographic columns and different elution methods on the separation of target objects, a more economical and environmentally friendly detection scheme is obtained. The results are as follows Figure 8 As shown, from Figure 8 It can be seen that the two characteristic peaks of the target substance appear almost simultaneously, which is not conducive to the actual sample detection and quantification. Therefore, this detection parameter condition is not suitable for the detection of samples with complex composition.

[0142] Comparative Example 3

[0143] The difference between Comparative Example 3 and Comparative Example 2 is that the mobile phase ratio in the detection parameters of ultra-high performance liquid chromatography is 20% water and 80% acetonitrile. Fig. 9 As shown, from Fig. 9 It can be seen that the two characteristic peaks of the target object still cannot be completely separated, which is not conducive to the detection and quantification of the sample. Therefore, this detection parameter condition is not suitable for the detection of samples with complex composition.

[0144] Comparative Example 4

[0145] The difference between Comparative Example 4 and Comparative Example 2 is that the mobile phase ratio in the detection parameters of ultra-high performance liquid chromatography is 30% water and 70% acetonitrile. Fig.10 As shown, Fig.10 This application relates to the superimposed chromatograms of the sweetener No. 4678 in the apple-pear flavor, menthol flavor and quality control samples. Fig.10It can be seen that the two characteristic peaks of the target substance in the quality control sample have been completely separated, and the peak time is 6.812min. However, in more complex samples, other substances co-elute, which will affect the quantitative results. Therefore, this detection parameter condition is not suitable for the detection of complex samples.

[0146] Comparative Example 5

[0147] The difference between Comparative Example 5 and Comparative Example 2 is that the mobile phase ratio in the detection parameters of ultra-high performance liquid chromatography is 43% water and 57% acetonitrile. Fig.11 As shown, Fig.11 This application relates to the superimposed chromatogram of the sweetener No. 4678 in the apple-pear flavored electronic atomization liquid sample and the quality control sample. Fig.11 It can be seen that the two characteristic peaks of the target substance in the quality control sample have been completely separated, and the peak time is 8.412min. However, in the more complex apple-pear flavored electronic atomization liquid sample, it will still be interfered by other substances, affecting the final quantitative results. Therefore, this detection parameter condition is not suitable for the detection of complex samples.

[0148] Comparative Example 6

[0149] The difference between Comparative Example 6 and Comparative Example 2 is that the mobile phase ratio in the detection parameters of ultra-high performance liquid chromatography is 95% 0.01% acetic acid and 5% methanol. Fig.12 As shown, Fig.12 This application relates to the superimposed chromatogram of the sweetener No. 4678 in the apple-pear flavored electronic atomization liquid sample and the quality control sample (100 mg / L). Fig.12 It can be seen that the peak time of the target substance in the quality control sample is 11.73min, but the two characteristic peaks are not completely separated. Similarly, the separation effect of the target substance in the more complex apple-pear flavored electronic atomization liquid sample is not good, which affects the final quantitative results. Therefore, this detection condition is not suitable for the detection and quantification of the target substance (sweetener No. 4678).

[0150] Comparative Example 7

[0151] The difference between Comparative Example 7 and Comparative Example 2 is that the mobile phase ratio in the detection parameters of ultra-high performance liquid chromatography is 95% 0.1% formic acid and 5% acetonitrile. Fig.13 As shown, Fig.13 This application relates to the chromatographic overlay comparison of sweetener No. 4678 in the apple-pear flavored electronic atomization liquid sample and the quality control sample (100 mg / L). Fig.13It can be seen that the peak time of the target in the quality control sample and the apple-pear flavored electronic atomization liquid sample is 10.67min, the two characteristic peaks are completely separated, and the peak shape is symmetrical, but the baseline is unstable, which will also affect the quantitative accuracy of the target. Therefore, this detection condition is not suitable for the detection and quantification of the target (sweetener No. 4678).

[0152] In summary, the detection method for N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide in oral consumer products provided in the present application has high sensitivity (detection limit: 3.5 μg / g, quantification limit: 11.8 μg / g), good accuracy and precision, short analysis time (11 minutes to complete separation and 13 minutes to end), strong anti-interference ability (symmetric peaks can be separated in complex samples), and takes into account the advantages of environmental protection and economy.

[0153] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide, characterized in that: include: A series of standard solutions are prepared, wherein different standard solutions contain different concentrations of the target substance, wherein the target substance is N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide; Using ultra-high performance liquid chromatography to test a series of standard solutions, and calculating a standard curve based on the test results; Pre-treating the sample to be tested to obtain a solution to be tested; The test solution is tested by ultra-high performance liquid chromatography, and the concentration of N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide in the test sample is determined according to the test result and the standard curve; The conditions of the ultra-high performance liquid chromatography include: the detection wavelength is 210-220nm.

2. The detection method according to claim 1, characterized in that: The method of pretreating the sample to be tested to obtain the solution to be tested includes: when the sample to be tested is liquid, obtaining a uniformly mixed sample to be tested, and placing the sample to be tested in a centrifuge tube; adding an extraction solvent to the centrifuge tube to fix the volume, then performing oscillation extraction for 20 to 30 minutes, taking the supernatant, and filtering to obtain the solution to be tested; when the sample to be tested is solid, obtaining the sample to be tested, crushing the sample to be tested, and placing the crushed sample to be tested in a centrifuge tube, adding an extraction solvent to the centrifuge tube to fix the volume, then performing oscillation extraction for 20 to 30 minutes, taking the supernatant, and filtering to obtain the solution to be tested.

3. The detection method according to claim 2, characterized in that: The extraction solvent is an organic solvent, preferably a pure solvent of ethanol, acetonitrile and methanol or a mixed solution of the organic solvent and water.

4. The detection method according to any one of claims 1 to 3, characterized in that: The chromatographic column used in the ultra-high performance liquid chromatography method is an octadecylsilane bonded silica gel chromatographic column or a chromatographic column equivalent to the octadecylsilane bonded silica gel chromatographic column; preferably, the chromatographic column is an end-capped chromatographic column.

5. The detection method according to claim 4, characterized in that: The conditions of the ultra-high performance liquid chromatography method include: column temperature of 20-35° C.; flow rate of 0.3 mL / min to 1 mL / min; injection volume of 2-10 μL.

6. The detection method according to claim 4, characterized in that: The conditions of the ultra-high performance liquid chromatography method include: the elution program is a gradient elution, and the gradient is as follows: 0-10min, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%; 10min, the volume fraction of mobile phase A is 5%, and the volume fraction of mobile phase B is 95%; 13min, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%; wherein mobile phase A is ultrapure water and mobile phase B is acetonitrile.

7. The detection method according to claim 4, characterized in that: The conditions of the ultra-high performance liquid chromatography method include: the chromatographic column is Poroshell 120EC-C18 or an equivalent chromatographic column.

8. The detection method according to claim 4, characterized in that: The conditions of the ultra-high performance liquid chromatography include: the chromatographic column is Agilent5HC-C18 chromatographic column or its equivalent chromatographic column, the elution procedure isocratic elution, the mobile phase A is ultrapure water, and the mobile phase B is acetonitrile: mobile phase A: mobile phase B = 3~4.5:5.5~7.

9. Use of the detection method according to any one of claims 1 to 8 in quality control of products containing N-(2-methylcyclohexyl)-2,3,4,5,6-pentafluoro-benzamide.

10. The use according to claim 9, characterized in that: The products include oral consumables.

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