Method for measuring loganic acid content in cosmetics

Abstract

The invention provides a method for determining the content of loganic acid in cosmetics, and relates to the technical field of cosmetic analysis. The determination method comprises the following steps: mixing a cosmetic sample with an extraction solvent which is a mixture of methanol, disodium ethylene diamine tetraacetate and sodium hydrogen sulfite, carrying out ultrasonic extraction and separation to obtain a supernatant, and carrying out elution purification and concentration through a solid-phase extraction column to obtain a sample to be detected; preparing a test solution, a standard stock solution and a mixed standard working solution; detecting the test solution by high performance liquid chromatography, drawing a standard curve to obtain a linear regression equation, and determining the content of loganic acid; the efficiency, accuracy, durability and the like of the method for testing the content of loganic acid in cosmetics are remarkably improved through a specific pretreatment method, an extraction solvent, a chromatographic column, the composition and proportion of a mobile phase, gradient elution conditions and the like.

Description

Technical Field

[0001] This invention relates to the field of cosmetic analysis technology, specifically to a method for determining the content of loganic acid in cosmetics. Background Technology

[0002] With the rapid development of the cosmetics industry, consumers are increasingly demanding higher efficacy and safety from products. Natural active ingredients, due to their gentle and highly effective properties, are widely used in various cosmetics. Loganic acid, a natural iridoid compound, is mainly derived from plant extracts such as Gentiana macrophylla and Cornus officinalis. It has significant anti-inflammatory, soothing, repairing, skin barrier-improving, and inflammatory factor-inhibiting effects. It can effectively relieve skin stinging and redness, improve sensitive skin, and also has certain antioxidant properties, which can help delay skin aging. Therefore, it is widely added to soothing and repairing, and anti-aging cosmetics, covering various dosage forms such as liquids, creams, and powders.

[0003] However, the content of loganic acid directly affects the efficacy and safety of cosmetics. Therefore, strictly controlling its content is of paramount importance, mainly for the following reasons: First, the need for efficacy stability. The skincare effects of loganic acid are significantly dose-dependent. Too low a content will prevent the product from achieving its claimed soothing and repairing effects, failing to meet consumer needs and impacting product reputation. Too high a content may disrupt the skin's physiological balance, causing adverse reactions such as skin irritation and redness, especially noticeable in people with sensitive skin. Second, the need for product quality uniformity. During cosmetic production, the actual content of loganic acid is prone to fluctuation due to factors such as raw material purity, extraction processes, and production control. Accurate measurement and control of its content can ensure the consistency of quality between different batches of products, meeting the standardized requirements of cosmetic production. Third, the need for safety supervision. Currently, relevant standards in the cosmetic industry do not have a clearly defined and unified limit on the amount of loganic acid added. However, to avoid potential safety risks, companies must control the loganic acid content within a reasonable and safe range through their own quality control, while also preventing false claims (such as claiming to contain loganic acid when it is not added or in extremely low amounts). Fourth, there is the need for raw material quality control. Loganic acid is mostly derived from plant extracts, and its content is greatly affected by factors such as the origin of the raw materials, the harvest time, and the extraction process. Accurately measuring the content of loganic acid in finished cosmetic products allows for reverse traceability of raw material quality, ensuring that the raw materials meet production requirements and guaranteeing product quality from the source.

[0004] Existing technologies also disclose methods for determining the content of loganic acid, but these are generally for determining the content of loganic acid in traditional Chinese medicinal materials. For example, Chinese patent application CN103175924A discloses a new method for simultaneously determining the content of multiple active ingredients in Cornus officinalis. This method uses high-performance liquid chromatography with diode array detection to simultaneously determine the content of seven components in Cornus officinalis from four major producing areas: loganin, gallic acid, 5-HMF, protocatechuic acid, monoglucoside, swertiamarin, and cornus officinalis glycoside. A Phenoemenex C18 polar chromatographic column is used, with acetonitrile and 0.1% formic acid solution as the mobile phase, and a flow rate of 1.0 mL / min. -1 Gradient elution, column temperature 30℃, detection wavelengths 220, 240, 260, 265 and 280 nm. "HPLC determination of 8-epicosuccinic acid content in Cistanche deserticola" (Pharmaceutical Analysis Impurities, 2006, 26(10), Yang Jianhua, Du Niansheng et al.) disclosed a method for determining the content of 8-epicosuccinic acid in different varieties of Cistanche deserticola by HPLC. The chromatographic conditions are as follows: Hypersil ODS-2 column (4.6 mm × 250 mm, 5 μm), mobile phase is acetonitrile-0.4% phosphoric acid (9:91), flow rate 1 mL·min -1 The column temperature was 25℃, and the detection wavelength was 235nm. However, existing technologies do not disclose methods for detecting the content of loganic acid in cosmetics.

[0005] However, the determination of loganic acid content in cosmetics currently faces the following technical challenges and limitations:

[0006] 1. Stability issues during extraction: Existing extraction methods mostly use direct ultrasonic extraction with methanol or ethanol aqueous solution. However, plant raw materials often contain residual endogenous enzymes. If effective enzyme inhibition measures are not taken during the extraction process, loganic acid will continue to undergo enzymatic hydrolysis in the extract, leading to lower test results that cannot truly reflect product quality.

[0007] 2. Severe interference from cosmetic matrix: Cosmetic formulations are complex, with lotions and creams containing large amounts of oils, emulsifiers, thickeners (such as carbomer and xanthan gum), and preservatives. Traditional liquid-liquid extraction or precipitation methods are insufficient to completely remove these matrix interferences. High molecular weight thickeners, in particular, not only easily form emulsion layers, making separation difficult, but may also adsorb target analytes or clog the chromatographic column, affecting the accuracy and reproducibility of the assay.

[0008] 3. Poor chromatographic retention: Loganiic acid is a highly polar compound. Current detection methods mostly employ traditional C18 reversed-phase columns with methanol / water or acetonitrile / water as the mobile phase. Due to its high polarity, loganiic acid exhibits very weak retention on C18 columns, making it highly susceptible to interference from solvent peaks and other polar impurities in the matrix. This results in peak tailing, poor resolution, and difficulty in achieving accurate quantification.

[0009] 4. Cumbersome pretreatment: Although some high-sensitivity methods (such as LC-MS / MS) are highly sensitive, pretreatment often requires complex solid phase extraction (SPE) steps, which are costly and time-consuming, making it difficult to meet the daily rapid quality inspection needs of cosmetic companies.

[0010] Therefore, developing a method for determining the content of loganic acid in cosmetics with high extraction efficiency, high separation, high sensitivity, and strong versatility, in order to achieve precise control of the loganic acid content and ensure the efficacy, safety, and quality uniformity of cosmetics, has become an urgent technical problem to be solved in the field of cosmetic testing. Summary of the Invention

[0011] To address the aforementioned problems, this invention provides a method for determining the content of loganic acid in cosmetics. By employing specific pretreatment methods, extraction solvents, chromatographic columns, mobile phase composition and ratio, and gradient elution conditions, the method significantly improves the efficiency, accuracy, and durability of the test.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides a method for determining the content of loganic acid in cosmetics, comprising the following steps: S1: Sample extraction: The cosmetic sample is mixed with the extraction solvent, which is a mixture of methanol, disodium ethylenediaminetetraacetate and sodium bisulfite. The mixture is extracted by ultrasonication, and the supernatant is separated. The supernatant is then purified by elution using a solid-phase extraction column and concentrated to obtain the sample to be tested. S2: Preparation of test solution: The sample to be tested is redissolved in an acetonitrile aqueous solution with a volume fraction of 90%-95%, filtered, and the test solution is obtained. S3: Preparation of standard stock solution: Accurately weigh loganic acid standard and dissolve it in acetonitrile aqueous solution with a volume fraction of 90%-95% to obtain a standard stock solution with a concentration of 100 μg / mL; S4: Preparation of mixed standard working solutions: Pipette 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 mL of the standard stock solution into 10 mL volumetric flasks, respectively, and dilute to the mark with 90%-95% acetonitrile aqueous solution. Shake well to obtain mixed standard working solutions with concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, and 50.0 μg / mL. S5: Detection: The test solution was detected by high performance liquid chromatography. Qualitative analysis was performed using the peak time and corresponding chromatogram of each component in the mixed standard solution. A standard curve was plotted using the mass concentration (X, μg / mL) corresponding to the peak area (Y) of each active ingredient in the mixed standard working solution. A linear regression equation was obtained to determine the content of loganic acid. The chromatographic conditions are as follows: Chromatographic column: Polar stationary phase chromatographic column; Mobile phase A: Ammonium acetate aqueous solution; Mobile phase B: Acetonitrile containing 0.05wt%-0.15wt% formic acid; Gradient elution, the elution procedure is as follows: .

[0013] Preferably, in step S1, the extraction solvent is ethylenediaminetetraacetic acid disodium (EDTA-2Na) and sodium bisulfite dissolved in a 65wt%-75wt% methanol aqueous solution, and the concentration of ethylenediaminetetraacetic acid disodium in the final solution is 8-12 mmol / L, and the concentration of sodium bisulfite is 0.2-0.8%, w / v.

[0014] More preferably, in step S1, the extraction solvent is ethylenediaminetetraacetic acid disodium (EDTA-2Na) and sodium bisulfite dissolved in a 70wt% methanol aqueous solution, and the concentration of ethylenediaminetetraacetic acid disodium in the final solution is 10 mmol / L and the concentration of sodium bisulfite is 0.5%, w / v.

[0015] Preferably, in step S1, the mass-to-volume ratio of the cosmetic sample to the extraction solvent is 1g:10-16mL; More preferably, in step S1, the mass-to-volume ratio of the cosmetic sample to the extraction solvent is 1g:12mL.

[0016] Preferably, in step S1, the ultrasonic extraction power is 280W-320W, the time is 10-25min, and the temperature is 20-30℃.

[0017] More preferably, in step S1, the ultrasonic extraction power is 300W, the time is 20min, and the temperature is 25℃.

[0018] Preferably, in step S1, the separation is centrifugation at a speed of 8000-10000 r / min for 5-10 min.

[0019] More preferably, in step S1, the separation is centrifugation at a speed of 9000 r / min for 8 min.

[0020] Preferably, in step S1, the packing material of the solid phase extraction column is a hydrophilic-lipophilic balanced material HLB.

[0021] More preferably, in step S1, the solid-phase extraction column elution purification includes the following steps: Activation: Activate the solid-phase extraction column sequentially with 3-10 mL of methanol and 3-10 mL of ultrapure water, and discard the eluent; Sample loading: Slowly pass the supernatant after centrifugation in step S1 through a solid-phase extraction column, controlling the flow rate at 0.5-1.5 mL / min; Washing: Wash the solid phase extraction column with 5-10 mL of ultrapure water to remove water-soluble impurities and discard the eluent; Elution: Elute the target substance with 3-10 mL of a 20%-30% (v / v) methanol aqueous solution and collect the eluent; Post-processing: The collected eluent was dried under nitrogen at 40°C and set aside for later use.

[0022] Preferably, in step S5, the chromatographic column is a HILIC column, 150 mm × 4.6 mm, 5 μm; Preferably, in step S5, the mobile phase A is an 8-12 mmol / L aqueous solution of ammonium acetate with a pH of 6-6.5; More preferably, in step S5, the mobile phase A is a 10 mmol / L ammonium acetate aqueous solution, and the pH is adjusted to 6.5 with ammonia. Preferably, in step S5, the mobile phase B is acetonitrile containing 0.1 wt% formic acid; Preferably, in step S5, the flow rate of the chromatographic conditions is 0.5-1.5 mL / min; Preferably, in step S5, the column temperature of the chromatographic conditions is 25-35℃; Preferably, in step S5, the detection wavelength of the chromatographic conditions is 230-250 nm; Preferably, in step S5, the injection volume under the chromatographic conditions is 8-12 μL; Preferably, in step S5, the gradient elution procedure is as follows: .

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. The method for determining the content of loganic acid in cosmetics provided by this invention, through specific pretreatment methods, extraction solvents, chromatographic columns, mobile phase composition and ratio, gradient elution conditions, etc., has pioneered a method for detecting the content of loganic acid in cosmetics, significantly improving the efficiency, accuracy, and durability of the test.

[0024] 2. This invention employs a composite extraction solvent system containing EDTA-2Na and sodium bisulfite, using a 70wt% methanol aqueous solution as the matrix, combined with the optimal concentrations of EDTA-2Na and sodium bisulfite. EDTA-2Na effectively reduces the adsorption of loganic acid by metal ions in the cosmetic matrix, while sodium bisulfite significantly inhibits the oxidative degradation of loganic acid. Combined with isothermal ultrasonic extraction and precise centrifugation parameters, efficient and complete extraction of loganic acid is achieved, significantly improving the extraction recovery rate while effectively avoiding degradation loss of loganic acid during the extraction process, thus ensuring the accuracy of the detection results.

[0025] 2. This invention uses a HILIC polar stationary phase column, combined with a specific mobile phase system and a precisely optimized gradient elution program. Formic acid can effectively improve the peak shape of loganic acid and inhibit its dissociation. The HILIC column has a stronger retention capacity for polar compounds, enabling baseline separation of structurally similar heterogeneous peaks (such as gentiopicrin and loganin) coexisting with loganic acid and cosmetics. The resolution is ≥1.5, and the tailing factor is controlled between 0.9 and 1.1. This effectively solves the quantitative deviation problem caused by insufficient resolution in existing methods. The detection precision RSD is ≤1.6%, and the quantitative accuracy is significantly improved.

[0026] 3. This invention clarifies the optimal ratio of sample to extraction solvent, key parameters for ultrasonic extraction, centrifugation, purification, and chromatographic detection. It eliminates the need to adjust core operating parameters for different cosmetic dosage forms such as aqueous solutions, creams, and powders, making it applicable to the detection of various cosmetics containing loganic acid. This solves the problems of poor versatility and cumbersome operation of existing methods. At the same time, it optimizes the detection process, shortens the detection cycle, eliminates the need for complex equipment, facilitates laboratory application, and reduces detection costs.

[0027] 4. This invention can accurately determine the content of loganic acid in cosmetics, effectively control the amount of loganic acid added within a reasonable range, avoid the product failing to achieve the claimed efficacy due to too low content, or causing adverse reactions such as skin irritation due to too high content. At the same time, the quality of raw materials can be traced back through finished product content detection, ensuring the uniformity of cosmetic quality, the stability of efficacy, and the safety of use from the source, which meets the standardized production and supervision requirements of the cosmetic industry. Attached Figure Description

[0028] Figure 1 This is the high-performance liquid chromatogram of Example 1. Detailed Implementation

[0029] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0030] Raw materials and instruments: Reagents: Methanol (chromatographic grade), disodium EDTA (analytical grade), sodium bisulfite (analytical grade), acetonitrile (chromatographic grade), ammonium acetate (chromatographic grade), formic acid (analytical grade). Standard: Loganilic acid standard (98%); Cosmetic samples to be tested: (1) Moisturizing and soothing water 1. Formula (100 g system) 88.0 g of deionized water, 5.0 g of butylene glycol, 3.0 g of 1,2-pentanediol, 2.0 g of 1,2-hexanediol, and 2.0 g of Gentiana macrophylla extract.

[0031] 2. Preparation process Weigh out deionized water and add butylene glycol, 1,2-pentanediol, and 1,2-hexanediol in sequence. Stir at medium speed for 5-10 minutes until completely clear and without layering. Add Gentiana macrophylla extract and stir for 5 minutes until homogeneous. Adjust the pH to 5-6, filter, let stand to defoam for 10-20 minutes, and then fill into containers.

[0032] (2) Moisturizing and soothing essence lotion The formula (per 100 g system) consists of the following ingredients by weight percentage: 1. Aqueous phase (81.0%) Deionized water 69.6%, glycerin 5.0%, 1,3-butanediol 3.0%, sodium hyaluronate 0.3%, dipotassium glycyrrhizate 0.2%, allantoin 0.2%, Gentiana macrophylla extract 2.0%, disodium EDTA 0.1%, carbomer 940 0.4%, triethanolamine 0.2%.

[0033] 2. Oil phase (16.6%) Squalane 4.0%, Caprylic / Capric Triglyceride 5.0%, Polydimethylsiloxane 3.0%, Glyceryl Stearate SE 2.5%, Cetearyl Alcohol 1.5%, Vitamin E Acetate 0.5%, Jojoba Oil 0.1%.

[0034] 3. Preservatives and fragrances (2.4%) p-Hydroxyacetophenone 0.5%, 1,2-hexanediol 1.5%, phenoxyethanol 0.3%, fragrance 0.1%.

[0035] Preparation process 1. Aqueous phase preparation Add deionized water, glycerol, 1,3-butanediol, and disodium EDTA to an aqueous phase vessel and heat to 75-80°C with stirring. Slowly add carbomer while stirring at high speed (500-800 rpm) until completely dissolved and free of particles.

[0036] Add sodium hyaluronate, dipotassium glycyrrhizate, and allantoin in sequence, and stir until completely dissolved.

[0037] Cool to 60℃, add Gentiana macrophylla extract, and stir well.

[0038] 2. Oil phase preparation Add squalane, caprylic / capric triglyceride, polydimethylsiloxane, glyceryl stearate (SE), cetearyl alcohol, vitamin E acetate, and jojoba oil to the oil phase pot, stir and heat to 75–80°C, until all oils / emulsifiers are completely melted and homogeneous.

[0039] 3. Emulsification Maintain the temperature of both the aqueous and oil phases at 75-80℃.

[0040] Slowly pour the oil phase into the aqueous phase while homogenizing at high speed (2000-3000 rpm) for 3-5 minutes to form a colostrum.

[0041] Continue stirring at low speed (300-500 rpm) and keep warm for 10 minutes to ensure complete emulsification and system stability.

[0042] 4. Cooling and subsequent addition Turn on the cooling and stir to lower the temperature to below 45°C.

[0043] Add triethanolamine, stir to neutralize carbomer, and adjust the pH to 5.5–6.5.

[0044] Add p-hydroxyacetophenone, 1,2-hexanediol, and phenoxyethanol in sequence, and stir until homogeneous.

[0045] Add flavoring and stir for 5 minutes.

[0046] 5. Maturation and Discharge Continue stirring and cooling to room temperature, then let stand for 24 hours to mature.

[0047] After testing pH, viscosity, and stability, the material is discharged and filled once it meets the requirements, thus completing the process.

[0048] instrument: 1260 Infinity II liquid chromatograph; F-060 Ultrasonic Cleaner; JC-WD-24 nitrogen blowing device.

[0049] Example 1 A method for determining the content of loganic acid in cosmetics, comprising the following steps: S1: Sample extraction: The cosmetic sample to be tested (moisturizing and soothing water) and the extraction solvent were mixed at a mass-volume ratio of 1g:12mL, and ultrasonic extraction was performed at a power of 300W, a time of 20min, and a temperature of 25℃. The mixture was then centrifuged at a speed of 9000r / min for 8min to obtain the supernatant. The supernatant was then eluted and purified by solid-phase extraction column and concentrated to obtain the sample to be tested. The extraction solvent is ethylenediaminetetraacetic acid disodium (EDTA-2Na) and sodium bisulfite dissolved in a 70wt% methanol aqueous solution. The concentration of ethylenediaminetetraacetic acid disodium in the final solution is 10 mmol / L, and the concentration of sodium bisulfite is 0.5%, w / v. The solid-phase extraction column elution and purification includes the following steps: Activation: Activate the solid-phase extraction column sequentially with 8 mL of methanol and 8 mL of ultrapure water, and discard the eluent; Sample loading: Slowly pass the supernatant after centrifugation in step S1 through a solid-phase extraction column, controlling the flow rate at 1 mL / min; Washing: Rinse the solid-phase extraction column with 8 mL of ultrapure water to remove water-soluble impurities and discard the eluent; Elution: Elute the target substance with 5 mL of 25% (v / v) methanol aqueous solution and collect the eluent; Post-processing: The collected eluent was dried under nitrogen at 40°C and set aside for later use.

[0050] S2: Preparation of test solution: The sample to be tested is redissolved in an aqueous solution of acetonitrile with a volume fraction of 95%, filtered, and the test solution is obtained. S3: Preparation of standard stock solution: Accurately weigh loganic acid standard and dissolve it in 95% acetonitrile aqueous solution to obtain a standard stock solution with a concentration of 100 μg / mL; S4: Preparation of mixed standard working solutions: Pipette 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 mL of the standard stock solution into 10 mL volumetric flasks, respectively, and dilute to the mark with 95% acetonitrile aqueous solution. Shake well to obtain mixed standard working solutions with concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, and 50.0 μg / mL. S5: Detection: The test solution was detected using high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: Chromatographic column: HILIC column, 150mm × 4.6mm, 5μm; Mobile phase A: 10 mmol / L ammonium acetate aqueous solution, pH adjusted to 6.5 with ammonia; Mobile phase B: Acetonitrile containing 0.1 wt% formic acid; Flow rate: 1 mL / min; Column temperature: 30℃; Detection wavelength: 240nm; Injection volume: 10 μL; Gradient elution, the elution procedure is shown in Table 1: Table 1

[0051] Chromatogram as shown Figure 1 As shown, the test method of the present invention exhibits good separation and good peak shape.

[0052] Qualitative analysis was performed using the peak times and corresponding chromatograms of each component in the mixed standard solution. A standard curve was plotted using the mass concentration (X, μg / mL) corresponding to the peak area (Y) of each active ingredient in the mixed standard working solution to obtain a linear regression equation, which was used to determine the content of loganic acid. The linear regression equation and correlation coefficient (R) were obtained. In addition, the limits of detection and limits of quantitation were calculated using the stepwise dilution method with a noise ratio of 3 times (S / N=3) and a noise ratio of 10 times (S / N=10), respectively. The results are shown in Table 2.

[0053] Table 2

[0054] As shown in Table 2, the test method of the present invention has good linearity, with R greater than 0.999. The method has high sensitivity and can fully meet the requirements for detecting the content of loganic acid in cosmetics without interference.

[0055] Example 2 Spiked recovery rate According to the preparation method of the moisturizing and soothing water described in this invention, loganic acid at low, medium, and high concentrations was added to a cosmetic blank matrix (the remaining components of the moisturizing and soothing water described in this invention, excluding Gentiana macrophylla extract) for spiked recovery experiments. Each spiked level was repeated six times, and the average recovery rate and relative standard deviation (RSD) were calculated. The results are shown in Table 3. The results show that the recovery rates at the three concentration levels were 95.8-101.5%, and the relative standard deviation (RSD) was 0.2-0.8%, indicating that the method has good accuracy and is suitable for the determination of loganic acid content in cosmetics.

[0056] Table 3

[0057] Example 3 Investigation of different flow rates Compared with Example 1, only the flow rate in step S5 was changed to 0.5 mL / min and 1.5 mL / min, while the rest remained the same as in Example 1. The results showed that the flow rate in the range of 0.5-1.5 mL / min had no significant effect on the results, and the obtained chromatograms had good resolution and good peak shape.

[0058] Example 4 Investigation at different column temperatures Compared with Example 1, only the column temperature in step S5 was changed to 25℃, 28℃, 32℃, and 35℃, while the rest remained the same as in Example 1. The results showed that the column temperature in the range of 25-35℃ had no significant effect on the results, and the obtained chromatograms had good resolution and peak shape.

[0059] Example 5 Investigation of different detection wavelengths Compared with Example 1, only the detection wavelength in step S5 was changed to 230nm, 235nm, 245nm, and 250nm, while the rest remained the same as in Example 1. The results showed that the detection wavelength in the range of 230-250nm had no significant effect on the results, and the obtained chromatograms had good separation and peak shape. However, the baseline was more stable when the wavelength was 240nm.

[0060] Example 6 Compared with Example 1, only the injection volume in step S5 was changed to 8 μL and 12 μL, while the rest remained the same as in Example 1. The results showed that the injection volume in the range of 8-12 μL had no significant effect on the results, and the obtained chromatograms had good resolution and peak shape.

[0061] Example 7 Examining different parameters in step S1: 7-1: S1: Sample extraction: The cosmetic sample to be tested (moisturizing and soothing water) and the extraction solvent were mixed at a mass-volume ratio of 1g:10mL, and ultrasonic extraction was performed at a power of 320W, a time of 10min, and a temperature of 20℃. The mixture was then centrifuged at a speed of 8000r / min for 10min to obtain the supernatant. The supernatant was then eluted and purified by solid-phase extraction column and concentrated to obtain the sample to be tested. The extraction solvent is ethylenediaminetetraacetic acid disodium (EDTA-2Na) and sodium bisulfite dissolved in a 65wt% methanol aqueous solution. The concentration of ethylenediaminetetraacetic acid disodium in the final solution is 12 mmol / L, and the concentration of sodium bisulfite is 0.2%, w / v. The rest is the same as in Example 1.

[0062] 7-2: S1: Sample extraction: The cosmetic sample to be tested (moisturizing and soothing water) and the extraction solvent were mixed at a mass-volume ratio of 1g:16mL, and ultrasonic extraction was performed at a power of 280W, a time of 25min, and a temperature of 30℃. The mixture was then centrifuged at a speed of 10000r / min for 5min to obtain the supernatant. The supernatant was then eluted and purified by solid-phase extraction column and concentrated to obtain the sample to be tested. The extraction solvent is ethylenediaminetetraacetic acid disodium (EDTA-2Na) and sodium bisulfite dissolved in a 75wt% methanol aqueous solution. The concentration of ethylenediaminetetraacetic acid disodium in the final solution is 8 mmol / L and the concentration of sodium bisulfite is 0.8%, w / v. The rest is the same as in Example 1.

[0063] The results showed that the obtained chromatograms had good resolution and good peak shape.

[0064] Example 8 Compared with Example 1, only the cosmetic sample to be tested was changed to a moisturizing and soothing essence lotion, while the rest was the same as in Example 1. The results showed that the obtained chromatogram had good separation and good peak shape.

[0065] Example 9 Methodological validation: (1) Precision A 10 μg / mL loganic acid solution was injected six times consecutively under high performance liquid chromatography (HPLC) conditions. The peak areas were then separated and measured. The relative standard deviation (RSD) of the peak area (n=6) was 1.23%, indicating that the system had good precision.

[0066] (2) Repeatability Six samples of cosmetics from the same batch were prepared in parallel according to the sample preparation method in Example 1. The samples were separated and determined by high performance liquid chromatography. The content of loganic acid in the samples was determined by the standard curve method. The calculated relative standard deviation (RSD) (n=6) was 1.42%, indicating that the method has good repeatability.

[0067] (3) Stability Loganic acid solutions with a concentration of 10 μg / mL were placed in an autosampler and analyzed at 0, 2, 6, 12, and 24 hours according to high performance liquid chromatography (HPLC) conditions. The peak areas of loganic acid at different times were calculated, and the relative standard deviations (RSDs) of the peak areas were 1.89%, indicating good stability.

[0068] Comparative Example 1 A method for determining the content of loganic acid in cosmetics, comprising the following steps: Compared with Example 1, only the extraction solvent in step S1 was changed to a methanol solution with a volume percentage of 80%, and the rest was the same as Example 1.

[0069] The results showed that when the extraction solvent was changed to 80% methanol (v / v), the solubility of loganic acid in the high-concentration organic solvent decreased due to its high polarity, and the high concentration of methanol extracted more lipid-soluble impurities. The test results indicated a significant decrease in the extraction recovery rate of loganic acid, increased baseline noise and more interfering peaks in the chromatogram, affecting the accuracy of quantification.

[0070] Comparative Example 2 A method for determining the content of loganic acid in cosmetics, comprising the following steps: Compared to Example 1, only the extraction solvent in step S1 was changed to: disodium ethylenediaminetetraacetate (EDTA-2Na) and sodium bisulfite dissolved in a 70wt% methanol aqueous solution, with the final solution containing 15 mmol / L of disodium ethylenediaminetetraacetate and 0.1% of sodium bisulfite, w / v. The rest remained the same as in Example 1.

[0071] The results showed that the chromatographic baseline drift was severe, the peak shape of loganic acid became wider, the detection limit deteriorated, and the sensitivity decreased.

[0072] Comparative Example 3 A method for determining the content of loganic acid in cosmetics, comprising the following steps: Compared with Example 1, only the chromatographic column in step S5 was changed to a Hypersil ODS-2 column (4.6 mm × 250 mm, 5 μm), and the rest was the same as in Example 1.

[0073] The results showed that the loganic acid chromatographic peak exhibited obvious tailing (tailing factor > 1.8) and insufficient separation from adjacent impurity peaks in the cosmetic matrix (R < 1.5), indicating a risk of co-elution.

[0074] Comparative Example 4 A method for determining the content of loganic acid in cosmetics, comprising the following steps: Compared with Example 1, only the mobile phase in step S5 was changed to acetonitrile-0.4% phosphoric acid (9:91), with isocratic elution, and the rest was the same as in Example 1.

[0075] The results showed that the loganic acid peak overlapped with the matrix interference peak, significantly reducing the resolution. Furthermore, the high proportion of aqueous phase led to prolonged analysis time, incomplete column cleaning, and retention time drift after continuous injection.

[0076] Comparative Example 5 A method for determining the content of loganic acid in cosmetics, comprising the following steps: Compared to Example 1, only the elution gradient program in step S5 was changed, as shown in Table 4 below: Table 4

[0077] The rest is the same as in Example 1.

[0078] The results showed that loganic acid eluted near the dead time (retention time <2 min), which could not be effectively separated from the solvent peak and polar impurities, resulting in inaccurate integration and quantification, and the method was completely ineffective.

[0079] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for determining the content of loganic acid in cosmetics, characterized in that, Includes the following steps: S1: Sample extraction: The cosmetic sample is mixed with the extraction solvent, which is a mixture of methanol, disodium ethylenediaminetetraacetate and sodium bisulfite. The mixture is extracted by ultrasonication, and the supernatant is separated. The supernatant is then purified by elution using a solid-phase extraction column and concentrated to obtain the sample to be tested. S2: Preparation of test solution: The sample to be tested is redissolved in an acetonitrile aqueous solution with a volume fraction of 90%-95%, filtered, and the test solution is obtained. S3: Preparation of standard stock solution: Accurately weigh loganic acid standard and dissolve it in acetonitrile aqueous solution with a volume fraction of 90%-95% to obtain a standard stock solution with a concentration of 100 μg / mL; S4: Preparation of mixed standard working solutions: Pipette 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 mL of the standard stock solution into 10 mL volumetric flasks, respectively, and dilute to the mark with 90%-95% acetonitrile aqueous solution. Shake well to obtain mixed standard working solutions with concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, and 50.0 μg / mL. S5: Detection: The test solution is detected by high performance liquid chromatography. Qualitative analysis is performed by the peak time and corresponding chromatogram of each component in the mixed standard solution. A standard curve is plotted by the mass concentration X corresponding to the peak area Y of each active ingredient in the mixed standard working solution to obtain the linear regression equation and determine the content of loganic acid. The chromatographic conditions are as follows: Chromatographic column: Polar stationary phase chromatographic column; Mobile phase A: Ammonium acetate aqueous solution; Mobile phase B: Acetonitrile containing 0.05wt%-0.15wt% formic acid; Gradient elution, the elution procedure is as follows: 。 2. The determination method according to claim 1, characterized in that, In step S1, the extraction solvent is ethylenediaminetetraacetic acid disodium salt and sodium bisulfite dissolved in a 65wt%-75wt% methanol aqueous solution. The concentration of ethylenediaminetetraacetic acid disodium salt in the final solution is 8-12 mmol / L, and the concentration of sodium bisulfite is 0.2-0.8%, w / v.

3. The determination method according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of the cosmetic sample to the extraction solvent is 1g:10-16mL; the ultrasonic extraction power is 280W-320W, the time is 10-25min, and the temperature is 20-30℃; the separation is centrifugation at a speed of 8000-10000r / min for 5-10min.

4. The determination method according to claim 1, characterized in that, In step S1, the packing material of the solid phase extraction column is HLB, a material with a hydrophilic-lipophilic balance.

5. The determination method according to claim 1, characterized in that, In step S1, the solid-phase extraction column is used for elution and purification. Includes the following steps: Activation: Activate the solid-phase extraction column sequentially with 3-10 mL of methanol and 3-10 mL of ultrapure water, and discard the eluent; Sample loading: Slowly pass the supernatant after centrifugation in step S1 through a solid-phase extraction column, controlling the flow rate at 0.5-1.5 mL / min; Washing: Wash the solid phase extraction column with 5-10 mL of ultrapure water to remove water-soluble impurities and discard the eluent; Elution: Elute the target substance with 3-10 mL of a 20%-30% (v / v) methanol aqueous solution and collect the eluent; Post-processing: The collected eluent was dried under nitrogen at 40°C and set aside for later use.

6. The determination method according to claim 1, characterized in that, In step S5, the chromatographic column is a HILIC column, 150 mm × 4.6 mm, 5 μm.

7. The determination method according to claim 1, characterized in that, In step S5, the mobile phase A is an aqueous solution of ammonium acetate at a concentration of 8-12 mmol / L and a pH of 6-6.

5.

8. The determination method according to claim 1, characterized in that, The mobile phase B is acetonitrile containing 0.1 wt% formic acid.

9. The determination method according to claim 1, characterized in that, In step S5, the flow rate of the chromatographic conditions is 0.5-1.5 mL / min; the column temperature of the chromatographic conditions is 25-35℃; the detection wavelength of the chromatographic conditions is 230-250 nm; and the injection volume of the chromatographic conditions is 8-12 μL.

10. The determination method according to claim 1, characterized in that, In step S5, the gradient elution procedure is as follows: 。

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