HPLC determination method for rapidly and efficiently extracting and detecting paeonol in cosmetics

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) determination method for rapidly and efficiently extracting and detecting paeonol in cosmetics, which comprises the following specific steps: S1, measuring to-be-detected cosmetic oil with the mass m, adding n-hexane with the volume V1, and carrying out vortex oscillation and ultrasonic treatment to obtain primary dispersion liquid; s2, adding the extracted primary dispersion liquid into methanol of which the volume is V2, and carrying out vortex oscillation again to obtain secondary dispersion liquid. According to the method, the content of paeonol in cosmetics can be efficiently and rapidly extracted and accurately measured, impurities are effectively removed through optimized solvent mixing and centrifugal filtration steps, the problem that an existing method is not suitable for cosmetic matrixes is solved, and the detection precision and accuracy are improved. According to the method, the reliability of a result is ensured by adopting a high performance liquid chromatography through an HPLC (High Performance Liquid Chromatography) chromatographic column, and an accurate determination result can be obtained even if a low-concentration phenolic active matter exists, so that the method is favorably applied to the detection of large-scale cosmetic samples.

Description

Technical Field

[0001] The invention relates to the field of cosmetic ingredient detection and analysis methods, and in particular to an HPLC determination method for rapidly and efficiently extracting and detecting paeonol in cosmetics. Background Art

[0002] Phenolic active ingredients are a class of compounds with multiple biological activities such as antioxidant, anti-inflammatory, and antibacterial activities, and are widely used in cosmetics. At the same time, due to the characteristics of phenolic substances being easily soluble in oil, they are often used in essential oil cosmetics. Therefore, in the field of cosmetic analysis technology, the determination of phenolic active ingredients is an important task.

[0003] At present, the method for detecting phenolic active ingredients in cosmetics mainly adopts high performance liquid chromatography (HPLC): This is a commonly used separation and analysis technology. Its principle is: using high performance liquid chromatography technology, under certain conditions, the compounds in the sample to be tested are separated, and then detected by a detector to finally obtain the content of the compound. High performance liquid chromatography has the advantages of high separation efficiency, fast analysis speed, and high detection sensitivity. However, when determining phenolic active ingredients, the traditional HPLC method often has problems such as low extraction efficiency and inability to be applied to a variety of phenolic substances.

[0004] Paeonol, as a Chinese herbal extract, has multiple effects such as antibacterial, anti-inflammatory and antioxidant, attracting the use of the cosmetics industry. However, the current common detection method for paeonol is to extract it from plant roots or Chinese medicine preparations. For cosmetic oil, its matrix contains a large amount of oil, and the extraction efficiency in traditional extraction reagents is low, so its test method is not applicable. The current method of using HPLC (high performance liquid chromatography) to determine phenolic active substances in cosmetics, although it has the advantages of high efficiency, accuracy and sensitivity, also has some shortcomings. These shortcomings are mainly reflected in the following aspects: the matrix of cosmetic samples is complex and often contains multiple components, which may interfere with the determination of phenolic active substances. Therefore, before the determination, the sample needs to be subjected to complex pretreatment, such as extraction, purification and other steps, which not only increases the difficulty of operation, but also may affect the accuracy of the determination results. In the HPLC determination process, the optimization of chromatographic conditions is crucial to the determination results. However, the types and contents of phenolic active substances in different cosmetic samples vary greatly, and the chromatographic conditions need to be optimized for different samples. This increases the workload and may also lead to inconsistency in the optimization results. The traditional HPLC analysis process usually takes a certain amount of time, which may lead to low detection efficiency and make it difficult to meet the needs of large-scale production or rapid detection.

[0005] For example, CN110763779A adopts the method of grinding and drying the sample and then adding methanol to extract it. In other fields, the extraction efficiency of phenolic active substances is also improved by improving the pretreatment extraction method or adjusting the chromatographic conditions, and cosmetic oils are complicated in matrix and have many additives, so these methods often have poor precision. The national standard "GB1886.233-2016 Food Safety National Standard Food Additive Vitamin E" uses saponification extraction to extract phenolic active substances in oil, but the process is time-consuming, the operation is cumbersome, the reagent consumption in the entire experimental process is large, the detection cycle is long, and because of multiple transfers, the extraction efficiency of this method is low, which affects the accuracy of the results. CN202310865656.9 uses enzymatic hydrolysis to extract tocopherols in vegetable oils, but the pretreatment process takes a long time, is troublesome to handle, and requires the consumption of a large amount of protease, which is costly. Therefore, the market is in urgent need of a detection method that can quickly and accurately detect paeonol in cosmetic oils. Summary of the invention

[0006] In view of the above problems, the present invention aims to provide a HPLC determination method for rapid and efficient extraction and detection of paeonol in cosmetics, which has good extraction effect, high detection accuracy and better precision.

[0007] To achieve the technical purpose, the scheme of the present invention is: a HPLC determination method for rapid and efficient extraction and detection of paeonol in cosmetics, the specific steps are as follows:

[0008] S1, measuring the mass m of the cosmetic oil to be tested, adding a volume V1 of n-hexane, vortexing and ultrasonicating to obtain a primary dispersion;

[0009] S2, adding methanol of volume V2 to the primary dispersion obtained by extraction, and vortexing again to obtain a secondary dispersion;

[0010] S3, adding V3 volume of methanol to the secondary dispersion again, and then centrifuging to obtain a centrifuge; using a pipette to reach into the lower layer of the centrifuge to take a sample, and filtering the lower layer of the centrifuge through a filter membrane to obtain a test solution;

[0011] S4. Using a HPLC column to detect the test solution and the paeonol standard solution, respectively, to obtain a paeonol standard curve, and finally to calculate the content X of paeonol in the test cosmetic oil.

[0012] Preferably, in step S4, the column temperature of the HPLC column is 25-35°C, the injection volume is 5-20 μL, the detection wavelength is 260-280 nm, and the mobile phase is divided into phase A and phase B, wherein phase A is a phosphoric acid aqueous solution with a mass percentage of 0.1-0.5%, and phase B is an acetonitrile solution; the flow rate of phase A is 0.8-1.5 mL / min, and the flow rate of phase B is 0.8-1.5 mL / min; the elution process adopts isocratic elution, the proportion of the mobile phase A is 10-40%, and the proportion of the mobile phase B is 60-90%.

[0013] Preferably, in step S1, the mass volume ratio of the cosmetic oil m to be tested and the added n-hexane V1 is 1:20 to 1:1; the mass m in step S1 is 0.2-1.0 g; the vortex oscillation time is 5-15 min, the ultrasound time is 10-30 min, and the ultrasound temperature is 25-35°C.

[0014] Preferably, the vortex oscillation time in step S2 is 2-8 min; the pore size of the filter membrane in step S3 is 0.45 μm; during the centrifugation process, the centrifuge speed is 5000-15000 r / min, and the centrifugation time is 4-6 min.

[0015] Preferably, a volume V3 of methanol is added to the secondary dispersion in step S3, wherein the ratio of the volume V1 of n-hexane to the total volume V2+V3 of the added methanol is in the range of 1:9 to 1:1; the mass volume ratio of the added cosmetic oil m to the total volume V1+V2+V3 of the added solvent is in the range of 1:40 to 1:5;

[0016] Preferably, the preparation process of the paeonol standard solution and the determination of the paeonol standard curve in step S4 are as follows:

[0017] Accurately weigh 25 mg of paeonol standard substance, place it in a 25 mL volumetric flask, add methanol to dissolve, continue to add methanol to the scale line, shake well, and obtain a 1000 mg / L paeonol standard solution;

[0018] Take 50 μL, 150 μL, 250 μL, 400 μL, and 500 μL of the 1000 mg / L paeonol standard stock solution respectively and place them in a 50 mL volumetric flask, add methanol to the mark to obtain paeonol standard solutions with corresponding concentrations of 1 mg / L, 3 mg / L, 5 mg / L, 8 mg / L, and 10 mg / L;

[0019] The paeonol standard solution was detected by high performance liquid chromatography, and the peak areas of each concentration were recorded to obtain a paeonol standard curve showing a linear relationship between paeonol concentration and peak area.

[0020] Preferably, the paeonol mass percentage content X is calculated according to the formula:

[0021]

[0022] Where: X——mass percentage content of paeonol in the cosmetic oil to be tested, %;

[0023] ρ——Paeonol concentration of the sample obtained from the paeonol standard curve, mg / L;

[0024] V——total volume of methanol and n-hexane added, V=V1+V2+V3, mL;

[0025] m——the mass of the cosmetic oil to be tested, g;

[0026] Preferably, the experimental result in step S4 is based on the arithmetic mean of the parallel determination results, and the relative difference of three independent determination results obtained under repeatability conditions is no more than 5% of the arithmetic mean.

[0027] The beneficial effects of the present invention are as follows: the method can efficiently and quickly extract and accurately determine the content of paeonol in cosmetics, effectively remove impurities through optimized solvent mixing and centrifugal filtration steps, solve the problem that the existing method cannot be applied to cosmetic matrices, and improve the precision and accuracy of detection. The reliability of the results is ensured by using high performance liquid chromatography through an HPLC column, and accurate measurement results can be obtained even for low concentrations of phenolic active substances, simplifying the equipment and operation, providing a scientific basis for cosmetic quality control, and being conducive to the application in the detection of large-scale cosmetic samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a picture of the standard curve of paeonol in the present invention;

[0029] Figure 2 is a high performance liquid chromatogram of the standard solution in the present invention;

[0030] Figure 3 It is a chromatogram of the high performance liquid of the test sample solution in the present invention. DETAILED DESCRIPTION

[0031] The following is combined with Figure 1-3 The present invention is further described in detail with reference to the accompanying drawings and specific embodiments.

[0032] The specific embodiment of the present invention is a HPLC determination method for rapid and efficient extraction and detection of paeonol in cosmetics, and the specific steps are as follows:

[0033] S1. Measure a mass m of the cosmetic oil to be tested, add a volume V1 of n-hexane, vortex oscillate, and perform ultrasound to obtain a primary dispersion; in step S1, the mass-to-volume ratio of the mass m of the cosmetic oil to be tested to the volume V1 of n-hexane added is 1:20 to 1:1, wherein the unit of m is g, and the unit of V1 is mL.

[0034] Hexane is a non-polar solvent that has good solubility for the oil components in cosmetics, and the solubility of phenolic active ingredients such as paeonol in oil is also relatively high. Vortex oscillation and ultrasonic treatment can accelerate the mixing of solvent and sample and improve extraction efficiency.

[0035] Specifically, the mass m is 0.2-1.0g, and the volume V1 of n-hexane added is 1-4mL; vortex oscillation promotes the mixing of sample and solvent by physical means, increases the contact area, and thus accelerates the transfer of solute from the sample matrix to the solvent. Taking advantage of the similar miscibility of n-hexane and oily compounds, a specific ratio of n-hexane and methanol mixture is selected to effectively disperse the analyte and improve the extraction efficiency of paeonol. If the amount of n-hexane added is too little, the extraction cannot completely extract the paeonol of the cosmetic oil, and the spike recovery rate is significantly reduced; if the amount of n-hexane added is too much, a large amount of n-hexane cannot be miscible with methanol, resulting in unclear quantitative volume, which will also seriously affect the spike recovery rate. In addition, the order of adding solvents will also affect its extraction efficiency.

[0036] At the same time, both the vortex oscillation time and the ultrasonic time have a significant effect on the extraction efficiency. If the vortex oscillation time and the ultrasonic time are short, the extraction rate is low and the detection accuracy cannot be met; if the vortex oscillation time and the ultrasonic time are too long, it is easy to produce too much invalid extraction time, affecting the detection efficiency. The vortex oscillation time is controlled to 5-15min. When ultrasonic waves propagate in liquids, cavitation effects, thermal effects and mechanical effects will occur. These effects can increase the frequency and speed of the movement of material molecules, increase the penetration of solvents, thereby accelerating the target components to enter the solvent and promote the extraction. Ultrasonic extraction has the advantages of high extraction efficiency, short extraction time and low solvent consumption. The ultrasonic time is controlled to 10-30min, and the ultrasonic temperature is controlled to 25-35℃.

[0037] S2, adding methanol of volume V2 to the primary dispersion obtained by extraction, and vortexing again to obtain a secondary dispersion;

[0038] Methanol is a polar solvent with good solubility for phenolic active substances. By adding methanol, the phenolic active substances in the primary dispersion can be further extracted to form a secondary dispersion. Specifically, the amount V2 of methanol added in step S2 is 2-6 mL, and the vortex oscillation time is 2-8 min.

[0039] S3, adding V3 volume of methanol to the secondary dispersion again, and then centrifuging to obtain a centrifuge; using a pipette to reach into the lower layer of the centrifuge to take a sample, and filtering the lower layer of the centrifuge through a filter membrane to obtain a test solution;

[0040] Adding methanol again can further dilute the solution, reduce the viscosity of the solution, and facilitate centrifugal separation. During the centrifugation process, due to the different densities and sedimentation rates of different components, the preliminary separation of the components can be achieved. The filter membrane is used to remove insoluble matter or impurities in the solution to obtain a pure test solution. Specifically, a volume of methanol V3 is added to the secondary dispersion of step S3, wherein the ratio of the volume V1 (ml) of n-hexane to the total volume V2+V3 (ml) of the added methanol is in the range of 1:9 to 1:1; the mass volume ratio of the added cosmetic oil m (g) to the total volume V1+V2+V3 (ml) of the added solvent is in the range of 1:40 to 1:5; wherein the pore size of the filter membrane is 0.45 μm; during the centrifugation process, the centrifuge speed is 5000-15000 r / min, and the centrifugation time is 4-6 min.

[0041] S4. Using a HPLC column to detect the test solution and the paeonol standard solution, respectively, to obtain a paeonol standard curve, and finally to calculate the content X of paeonol in the test cosmetic oil.

[0042] HPLC (High Performance Liquid Chromatography) is a separation technique based on the difference in the partition coefficient of solutes between the stationary phase and the mobile phase. Different components in the test solution can be separated by an HPLC column and detected by a detector. The paeonol standard curve is obtained by detecting a series of paeonol standard solutions of known concentrations, which is used to quantitatively calculate the paeonol content in the test solution.

[0043] Specifically, in step S4, the column temperature of the HPLC chromatographic column is 25-35°C, and the column temperature affects the partition coefficient of the solute between the stationary phase and the mobile phase, thereby affecting the separation and retention time of the chromatographic peak. Appropriate column temperature can improve the separation efficiency of the chromatographic column and reduce peak broadening. The column temperature is set within the range of 25-35°C, which helps to maintain the stability and separation effect of the chromatographic column, while avoiding solvent volatilization or chromatographic column damage caused by excessive temperature. The injection volume is 5-20 μL, and the injection volume determines the amount of sample entering the chromatographic column, thereby affecting the intensity and sensitivity of the chromatographic peak. The injection volume is set within the range of 5-20 μL, which can ensure sufficient sample volume to obtain a clear chromatographic peak, while avoiding peak distortion or column efficiency reduction caused by overload. The detection wavelength is 260-280nm, and the detection wavelength should be selected at the wavelength where the analyte (such as paeonol) has the maximum absorption peak to improve the sensitivity and accuracy of detection. The detection wavelength is set within the range of 260-280 nm, which is based on the fact that the paeonol active substance has strong ultraviolet absorption in this wavelength region, and helps to achieve high-sensitivity detection.

[0044] The mobile phase is divided into phase A and phase B. The composition and flow rate of the mobile phase directly affect the retention time and separation degree of the solute on the chromatographic column. The chromatographic separation effect can be optimized by adjusting the polarity and flow rate of the mobile phase.

[0045] Phase A is a 0.1-0.5% by mass phosphoric acid aqueous solution, and phase B is an acetonitrile solution;

[0046] The flow rate of phase A is 0.8-1.5mL / min, and the flow rate of phase B is 0.8-1.5mL / min; by adjusting their ratio and flow rate, effective separation of solutes of different polarities can be achieved. The elution process adopts isocratic elution, and the isocratic elution method (that is, the mobile phase ratio and flow rate remain unchanged during the elution process) simplifies the operation process and helps to obtain a stable chromatogram. The proportion of mobile phase A is 10-40%, and the proportion of mobile phase B is 60-90%. The ratio of phase A and phase B in the mobile phase determines the polarity and elution strength of the mobile phase, which in turn affects the retention time and separation degree of the solute. Setting the proportion of phase A to 10-40% and the proportion of phase B to 60-90% can optimize the polarity characteristics of paeonol to ensure that they can be well separated and detected on the chromatographic column.

[0047] The preparation process of paeonol standard solution and the determination of paeonol standard curve are as follows:

[0048] Accurately weigh 25 mg of paeonol standard substance, place it in a 25 mL volumetric flask, add methanol to dissolve, continue to add methanol to the scale line, shake well, and obtain a 1000 mg / L paeonol standard solution;

[0049] Take 50 μL, 150 μL, 250 μL, 400 μL, and 500 μL of the 1000 mg / L paeonol standard stock solution respectively and place them in a 50 mL volumetric flask, add methanol to the mark to obtain paeonol standard solutions with corresponding concentrations of 1 mg / L, 3 mg / L, 5 mg / L, 8 mg / L, and 10 mg / L;

[0050] The paeonol standard solution was detected by high performance liquid chromatography, and the peak areas of each concentration were recorded to obtain a paeonol standard curve showing a linear relationship between paeonol concentration and peak area.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) Easy operation. Compared with other pre-treatment methods such as enzymatic hydrolysis, saponification, and direct extraction, the present invention adopts an extraction method. While not reducing the sample recovery rate, the entire operation process is simple and easy to use, effectively reducing the detection cycle.

[0053] (2) Low cost. Compared with other pretreatment methods, the present invention only requires two common pretreatment reagents, which effectively reduces the cost.

[0054] (3) It fills the gap in the detection of phenolic active ingredients in cosmetics. Currently, there is no detection method for phenolic active ingredients in cosmetics. Since the amount of active ingredients added in cosmetics is relatively low, their accurate and efficient determination is somewhat challenging.

[0055] When the determination method is used, the ratio of n-hexane to total methanol is preferably 2:8, and the mass volume ratio of the analyte to the solvent is preferably 0.5 g:10 mL. As provided in the present invention, a specific embodiment - Embodiment 1, the three cosmetic oils in Embodiment 1 are cosmetic oil products to which paeonol is added, and the specific method of Embodiment 1 is:

[0056] (1) Weigh two portions of 0.5 g (accurate to 0.0001 g) of the cosmetic oil sample to be tested and place them in a 30 mL centrifuge tube, add 2 mL of n-hexane, vortex and oscillate for 10 min, and then place them in an ultrasonic cleaner and sonicate at 30 ° C for 20 min to obtain a primary dispersion;

[0057] (2) Add 4 mL of methanol to the primary dispersion and vortex again for 5 min to obtain a secondary dispersion;

[0058] (3) Add 4 mL of methanol to the secondary dispersion, shake well, and centrifuge at 10,000 r / min for 5 min; use a pipette to reach into the lower layer to take a sample, and filter through a 0.45 μm filter membrane to obtain a test solution;

[0059] (4) The test liquid is detected by high performance liquid chromatography to obtain the corresponding content of paeonol; the column temperature of the HPLC column is 30°C, the injection volume is 10 μL, the detection wavelength is 275 nm, and the mobile phase is divided into phase A and phase B, wherein phase A is a 0.1% phosphoric acid aqueous solution and phase B is an acetonitrile solution; the flow rate of phase A and phase B is 1.0 mL / min; the elution process adopts isocratic elution, and the ratio of mobile phase A to mobile phase B is 20% and 80%.

[0060] (5) Weigh 25 mg (accurate to 0.0001 g) of paeonol standard substance and place it in a 25 mL volumetric flask. Add an appropriate amount of methanol to dissolve it. Continue to add methanol to the scale line and shake well to obtain a 1000 mg / L paeonol standard solution.

[0061] (6) Take 50 μL, 150 μL, 250 μL, 400 μL, and 500 μL of the 1000 mg / L paeonol standard stock solution respectively and place them in a 50 mL volumetric flask, add methanol to the scale, and obtain paeonol standard solutions with corresponding concentrations of 1 mg / L, 3 mg / L, 5 mg / L, 8 mg / L, and 10 mg / L. It can be seen that the standard curve of p-hydroxyacetophenone has good linearity in the concentration range of 1.0 to 20 mg / L, and R2 is 0.999.

[0062] (7) Determination: Under the reference chromatographic conditions, the standard solution and the sample were measured respectively, the area of ​​the peak was recorded, and the content of paeonol in the sample was calculated according to the formula.

[0063] (8) Calculation of results: The experimental results are based on the arithmetic mean of the parallel determination results. The relative difference of three independent determination results obtained under repeatability conditions is no more than 5% of the arithmetic mean, which improves the accuracy and reliability of the experimental results and ensures the stability and repeatability of the experimental method.

[0064] Finally, the mass percentage X of paeonol can be calculated according to the formula:

[0065]

[0066] Where: X——the content of paeonol in the cosmetic oil to be tested, %;

[0067] ρ——Paeonol concentration of the sample obtained from the paeonol standard curve, unit: mg / L;

[0068] V——total volume of methanol and n-hexane added, V=V1+V2+V3, unit: mL;

[0069] m——the mass of the cosmetic oil to be tested, in g;

[0070] The above method was used to determine the content of paeonol in different cosmetic oils, and a high recovery rate was achieved, indicating a good extraction effect.

[0071] As shown in Table 1, the experimental results show that within the concentration range of 1-10 mg / L, the linearity between the paeonol content in the sample tube and the peak area is good, the correlation coefficient is greater than 0.999, the test results are accurate, and the error is small; Figure 1 Shown is a picture of the standard curve of paeonol.

[0072] Table 1: Standard curve of paeonol

[0073]

[0074] As shown in Table 2, the experimental results show that the analyte was spiked with low, medium and high concentrations within the curve range, and at least 2 sets of parallel tests were performed for each concentration. The overall recovery rate results were all within 5%, and the test results were good. Figure 2 Shown is the HPLC chromatogram of the standard.

[0075] Table 2: Determination results of paeonol content in three cosmetic oils

[0076]

[0077]

[0078] In order to explore the precision of the detection method of paeonol in cosmetic oil provided by the present invention, the method of Example 1 was used to repeat the experiment on the sample, as shown in Table 3 below. The experimental results show that the extraction efficiency of paeonol can be >90%, the spike recovery rate can be >90%, and the RSD can be <5% by using the present invention to extract common cosmetic oils on the market. The detection accuracy is high and the precision is strong. Figure 3 The HPLC chromatogram of the test sample.

[0079] Table 3: Determination results of paeonol content in cosmetic oils at various precisions

[0080]

[0081]

[0082] In order to explore the detection limit and quantification limit of the detection method of paeonol in cosmetic oil provided by the present invention, a signal-to-noise ratio test was performed on a signal of a certain concentration, the signal-to-noise ratio S / N, the detection limit (LOD) was three times the test concentration divided by the signal-to-noise ratio, that is, LOD=3*S / N; the quantification limit (LOQ) was ten times the test concentration divided by the signal-to-noise ratio, that is, LOQ=10*S / N. The sample was tested using the method of Example 1, and the detection limit is shown in Table 4, and the quantification limit is shown in Table 5.

[0083] Table 4: Detection limits of paeonol in cosmetic oils

[0084]

[0085] Table 5: Quantitative limits of paeonol in cosmetic oils

[0086]

[0087] In order to verify the effectiveness of the determination method, the present application also provides comparative examples 1-3 for comparing the effects of different experimental conditions on the experimental results. Among them, comparative example 1 and example 1 use different extraction methods; comparative examples 2-3 and example 1 use the same extraction method.

[0088] The scheme of comparative example 1 is as follows: a sample with known paeonol content is processed, a certain mass of sample is weighed and placed in a colorimetric tube, methanol is added to the scale line, the volumetric bottle stopper is plugged, shaken, its mass is weighed, and then ultrasonic treatment is performed under low temperature conditions. After ultrasonic treatment, its mass is weighed again, and the mass lost compared with the first weighing is supplemented with methanol: then shaken, and the solution in the volumetric bottle is filtered with an organic microporous filter membrane with a pore size of 0.22um, the filtrate is placed in another volumetric bottle, and methanol is added to dilute to the scale, shaken, and the sample to be tested is obtained. Filtration is to remove impurities to prevent clogging of the chromatographic column after injection, so that paeonol cannot pass through the chromatographic column or cause the peak time to change, thereby affecting the test results. Table 6 shows the results of comparative example 1 on the sample processing. It can be seen from the comparison test results that due to the different matrix of the sample, methanol cannot completely extract the paeonol in the cosmetic oil, and there is an incomplete extraction and poor precision.

[0089] Table 6: Results of sample treatment in Comparative Example 1

[0090] serial number Determination results of paeonol content in cosmetic oil (unit: %) Precision 1 0.0073 Precision 2 0.0075 Precision 3 0.0088 Precision 4 0.0064 Precision 5 0.0077 Precision 6 0.0092 Precision 7 0.0057 Precision 8 0.0093 Precision 9 0.0077 Precision 10 0.0082 average value 0.0078 RSD (Relative Standard Deviation) 14.90

[0091] The difference between Example 2 and Example 1 is that the proportion of the extraction solvent added in steps 1-3 is different. The amount of n-hexane added in the first step was changed to 1mL and 5mL, respectively, and the samples were extracted and tested as Example 2-1 and Example 2-2. The reproducibility and precision of the test results of Example 2-1 are shown in Table 7. When the amount of n-hexane added is 1mL, the test sample cannot be completely extracted, and the extraction recovery rate is not ideal, while Example 1 can effectively improve the extraction rate of paeonol; when the amount of n-hexane added is too much, due to the weak solubility of n-hexane in methanol and a certain solubility in oil, the sample cannot be completely extracted, the solvent and the sample cannot be completely stratified, and the purpose of extraction cannot be achieved.

[0092] Table 7: Measurement results when the amount of n-hexane added was 1 mL

[0093] serial number Determination results of paeonol content in cosmetic oil (unit: %) Precision 1 0.0093 Precision 2 0.0088 Precision 3 0.0103 Precision 4 0.0077 Precision 5 0.0098 Precision 6 0.0094 Precision 7 0.0089 Precision 8 0.0093 Precision 9 0.0107 Precision 10 0.0065 average value 0.0091 RSD (Relative Standard Deviation) 13.46

[0094] The difference between Comparative Example 3 and Example 1 is that the ultrasonic time in step 1-3 is different. The ultrasonic time of the first step in Example 1 was changed to 10 minutes and 30 minutes, respectively, and recorded as Comparative Example 3-1 and Comparative Example 3-2. The reproducibility and precision of the test results are shown in Tables 8 and 9. When the ultrasonic extraction time is short, the test sample cannot be completely extracted, and the extraction recovery rate is not ideal; when the time is increased, the recovery rate of the test results is not much different from that of Example 1, indicating that the sample has been completely extracted under the time conditions of Example 1, and increasing the ultrasonic time has no effect on the test results, only increasing the time cost of the test.

[0095] Table 8: Test results when ultrasound time is reduced

[0096]

[0097]

[0098] Table 9: Test results with increasing ultrasound time

[0099] serial number Determination results of paeonol content in cosmetic oil (unit: %) Precision 1 0.0102 Precision 2 0.0104 Precision 3 0.0103 Precision 4 0.0098 Precision 5 0.0096 Precision 6 0.0098 Precision 7 0.0099 Precision 8 0.0101 Precision 9 0.0100 Precision 10 0.0098 average value 0.0100 RSD (Relative Standard Deviation) 2.56

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any slight modification, equivalent substitution and improvement made to the above embodiment based on the technical essence of the present invention should be included in the protection scope of the technical solution of the present invention.

Claims

1. A rapid and efficient HPLC method for extracting and detecting paeonol in cosmetics, characterized in that: The specific steps are as follows: S1, measuring the mass m of the cosmetic oil to be tested, adding a volume V1 of n-hexane, vortexing and ultrasonicating to obtain a primary dispersion; S2, adding methanol of volume V2 to the primary dispersion obtained by extraction, and vortexing again to obtain a secondary dispersion; S3, adding V3 volume of methanol to the secondary dispersion again, and then centrifuging to obtain a centrifuge; using a pipette to reach into the lower layer of the centrifuge to take a sample, and filtering the lower layer of the centrifuge through a filter membrane to obtain a test solution; S4. Using a HPLC column to detect the test solution and the paeonol standard solution, respectively, to obtain a paeonol standard curve, and finally to calculate the content X of paeonol in the test cosmetic oil.

2. The HPLC determination method for rapid and efficient extraction and detection of paeonol in cosmetics according to claim 1, characterized in that: In step S4, the column temperature of the HPLC chromatographic column is 25-35° C., the injection volume is 5-20 μL, the detection wavelength is 260-280 nm, and the mobile phase is divided into phase A and phase B, wherein phase A is a phosphoric acid aqueous solution with a mass percentage of 0.1-0.5%, and phase B is an acetonitrile solution; the flow rate of phase A is 0.8-1.5 mL / min, and the flow rate of phase B is 0.8-1.5 mL / min; the elution process adopts isocratic elution, the proportion of the mobile phase A is 10-40%, and the proportion of the mobile phase B is 60-90%.

3. The HPLC determination method for rapid and efficient extraction and detection of paeonol in cosmetics according to claim 2, characterized in that: In step S1, the mass volume ratio of the cosmetic oil m to be tested and the added n-hexane V1 is 1:20 to 1:1; the mass m in step S1 is 0.2-1.0 g; the vortex oscillation time is 5-15 min, the ultrasound time is 10-30 min, and the ultrasound temperature is 25-35°C.

4. The HPLC determination method for rapid and efficient extraction and detection of paeonol in cosmetics according to claim 3, characterized in that: The vortex oscillation time in step S2 is 2-8 minutes; the pore size of the filter membrane in step S3 is 0.45 μm; during the centrifugation process, the centrifuge speed is 5000-15000 r / min, and the centrifugation time is 4-6 minutes.

5. The HPLC determination method for rapid and efficient extraction and detection of paeonol in cosmetics according to claim 3, characterized in that: A volume V3 of methanol is added to the secondary dispersion in step S3, wherein the ratio of the volume V1 of n-hexane to the total volume V2+V3 of the added methanol is in the range of 1:9 to 1:1; the mass volume ratio of the added cosmetic oil m to the total volume V1+V2+V3 of the added solvent is in the range of 1:40 to 1:5;.

6. The HPLC determination method for rapid and efficient extraction and detection of paeonol in cosmetics according to claim 3, characterized in that: The preparation process of the paeonol standard solution and the determination of the paeonol standard curve in step S4 are as follows: Accurately weigh 25 mg of paeonol standard substance, place it in a 25 mL volumetric flask, add methanol to dissolve, continue to add methanol to the scale, shake well, and obtain a 1000 mg / L paeonol standard solution; Take 50 μL, 150 μL, 250 μL, 400 μL, and 500 μL of the 1000 mg / L paeonol standard stock solution respectively and place them in a 50 mL volumetric flask, add methanol to the mark to obtain paeonol standard solutions with corresponding concentrations of 1 mg / L, 3 mg / L, 5 mg / L, 8 mg / L, and 10 mg / L; The paeonol standard solution was detected by high performance liquid chromatography, and the peak areas of each concentration were recorded to obtain a paeonol standard curve showing a linear relationship between paeonol concentration and peak area.

7. The HPLC determination method for rapid and efficient extraction and detection of paeonol in cosmetics according to claim 6, characterized in that: The mass percentage content X of paeonol can be calculated according to the formula: Where: X——mass percentage content of paeonol in the cosmetic oil to be tested, %; ρ——Paeonol concentration of the sample obtained from the paeonol standard curve, mg / L; V——total volume of methanol and n-hexane added, V=V1+V2+V3, mL; m——The mass of the cosmetic oil to be tested, g.

8. The HPLC determination method for rapid and efficient extraction and detection of paeonol in cosmetics according to claim 7, characterized in that: The experimental result in step S4 is based on the arithmetic mean of the parallel determination results, and the relative difference of three independent determination results obtained under repeatability conditions is no more than 5% of the arithmetic mean.

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