Method for detecting components of a cosmetic fluid and method for detecting the penetration properties of a cosmetic fluid

Abstract

The application relates to a component detection method of a cosmetic liquid and a permeability detection method of the cosmetic liquid, and the component detection method comprises the following steps: adding a protein precipitant to purify the cosmetic liquid; performing liquid chromatography-tandem mass spectrometry on the purified cosmetic liquid to obtain chromatographic peak response values of nicotinamide, ascorbic acid and VC ethyl ether; wherein, the positive ion and the negative ion are alternately ionized in the mass spectrometry, and multiple reaction monitoring is performed on the nicotinamide, the ascorbic acid and the VC ethyl ether; and the concentrations of the nicotinamide, the ascorbic acid and the VC ethyl ether in the cosmetic liquid are obtained according to the chromatographic peak response values. The method improves the detection precision of low-concentration and multi-target component.

Description

Technical Field

[0001] This application relates to the field of cosmetic technology, and in particular to methods for detecting the components of beauty serums and methods for detecting the permeability of beauty serums. Background Technology

[0002] Niacinamide, 3-O-ethyl-L-ascorbic acid (VC ethyl ether), and tranexamic acid are widely used whitening and anti-inflammatory active ingredients in cosmetics. They are often added to face creams, serums, lotions, and the rapidly developing beauty mists to synergistically exert whitening, antioxidant, and anti-melanin production effects. To evaluate the transdermal absorption capacity of these functional ingredients in practical applications, in vitro transdermal assays have become an important efficacy evaluation method. However, due to the low concentration of each target functional ingredient, simultaneous detection of multiple target functional ingredients suffers from poor sensitivity and low accuracy. Summary of the Invention

[0003] Therefore, it is necessary to provide a method for detecting the components of a beauty serum and a method for detecting the permeability of a beauty serum, so as to improve the detection accuracy of low-concentration, multi-target substances.

[0004] One aspect of this application provides a method for detecting the components of a beauty serum, the beauty serum comprising niacinamide, tranexamic acid, and VC ethyl ether, the method comprising the following steps:

[0005] The beauty essence is purified by adding a protein precipitant;

[0006] The purified beauty serum was subjected to liquid chromatography-tandem mass spectrometry (LC-MS / MS) to obtain the chromatographic peak response values ​​of nicotinamide, tranexamic acid, and VC ethyl ether; wherein...

[0007] The nicotinamide, tranexamic acid, and VC ethyl ether were subjected to multiple reaction monitoring in the mass spectrometer using alternating positive and negative ion ionization modes.

[0008] The concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the beauty serum were determined based on the chromatographic peak response values.

[0009] The above method targets beauty serums containing three active ingredients: nicotinamide, tranexamic acid, and VC ethyl ether. It simultaneously detects these three ingredients using liquid chromatography-tandem mass spectrometry (LC-MS / MS). This method removes matrix interferences from the beauty serum by adding a protein precipitant, reducing ion effects and chromatographic interference. In mass spectrometry, the method employs alternating positive and negative ion ionization modes. Multiple reaction monitoring (MRM) is performed on nicotinamide and tranexamic acid in positive ion ionization mode, and on VC ethyl ether in negative ion ionization mode. This achieves efficient separation and stable response of three compounds with different polarities and ionization characteristics—nicotinamide, VC ethyl ether, and tranexamic acid—within the same system, solving the problem of traditional methods being unable to accurately determine these three active ingredients simultaneously. Furthermore, this method lowers the detection limit, effectively meeting the detection needs when the concentration of active ingredients in the beauty serum is extremely low, and improving the detection accuracy for low-concentration, multi-target substances.

[0010] In some embodiments, the multiple reaction monitoring includes the following steps:

[0011] Monitoring the transitions of nicotinamide from parent ion m / z 123 to daughter ion m / z 78 and / or from parent ion m / z 123 to daughter ion m / z 80; and / or,

[0012] For tranexamic acid, monitor the transitions from parent ion m / z 158 to daughter ion m / z 67 and / or the transitions from parent ion m / z 158 to daughter ion m / z 95; and / or; and / or,

[0013] For VC ethyl ether, monitor the transitions from the parent ion m / z 203 to the daughter ion m / z 85 and / or the transitions from the parent ion m / z 203 to the daughter ion m / z 131.

[0014] In some embodiments, the protein precipitant comprises acetonitrile at a temperature of 0°C to 4°C.

[0015] In some embodiments, the operating conditions of the liquid chromatography include:

[0016] The chromatographic column is a Waters BEH C18 column; and / or,

[0017] Column temperature is 35°C to 45°C; and / or,

[0018] The flow rate of the mobile phase is 0.3 mL / min to 0.6 mL / min.

[0019] In some embodiments, the mobile phase of the liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of acetic acid and mobile phase B is a methanol solution of acetic acid;

[0020] Optionally, the liquid chromatography performs gradient elution, which includes the following steps:

[0021] During the time period from 0 to t1, where t1 is 0 min to 1.0 min, the volume percentage of mobile phase A in the mobile phase is controlled to be 90% to 98%, and the volume percentage of mobile phase B in the mobile phase is controlled to be 2% to 10%.

[0022] During the time interval from t2 to t3, t2 is 1.0 min to 2.5 min and t3 is 2.5 min to 4.0 min. The volume percentage of mobile phase A in the mobile phase is controlled to be 5% to 15%, and the volume percentage of mobile phase B in the mobile phase is controlled to be 85% to 95%.

[0023] During the time interval from t4 to t5, t4 is 4.0 min ~ 4.1 min and t5 is 4.1 min ~ 7.0 min. The volume percentage of mobile phase A in the mobile phase is 90% ~ 98%, and the volume percentage of mobile phase B in the mobile phase is 2% ~ 10%.

[0024] In some embodiments, the operating conditions of the mass spectrometer include:

[0025] Waters H-Class LC coupled with TQS-micro triple quadrupole mass spectrometry; and / or,

[0026] The temperature of the ion source is 140°C to 180°C; and / or,

[0027] The ionization mode is an alternating ionization mode of ESI positive ions and ESI negative ions; and / or,

[0028] The solvent removal temperature is 400°C ~ 550°C; and / or,

[0029] The flow rate of the desolvation gas is 800 L / h ~ 1100 L / h; and / or,

[0030] The flow rate of the conical orifice gas is 1 L / h ~ 20 L / h.

[0031] In some embodiments, the beauty serum contains 0.0001% to 5% niacinamide, 0.0001% to 5% tranexamic acid, and 0.0001% to 5% VC ethyl ether by mass percentage; and / or,

[0032] The beauty essence is selected from beauty atomizing liquid, beauty spray, toner, beauty serum, beauty gel or beauty cream.

[0033] A second aspect of this application provides a method for testing the permeability of a beauty serum, the beauty serum comprising niacinamide, tranexamic acid, and VC ethyl ether, the method comprising the following steps:

[0034] An in vitro transdermal test was conducted on the beauty serum, and the permeate was collected at different time points;

[0035] The concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the permeate are determined using the method described in the first aspect.

[0036] In some embodiments, the method for testing the permeability of the beauty serum includes the following steps:

[0037] The ex vivo transdermal test includes the following steps:

[0038] The excised animal skin is fixed between the supply chamber and the receiving chamber of the Franz diffusion cell, with the keratin layer of the animal skin facing the supply chamber and the dermis layer of the animal skin facing the receiving chamber.

[0039] Add the recipient fluid to the receiving chamber;

[0040] The beauty solution is added to the supply chamber and comes into contact with the stratum corneum of the animal skin to achieve transdermal penetration, and the permeated solution is obtained in the receiving chamber.

[0041] In some embodiments, the recipient fluid comprises at least one of a mixture of physiological saline and ethanol, or a mixture of phosphate-buffered saline and ethanol; and / or,

[0042] The temperature of the receptor solution is 30°C to 37°C; and / or,

[0043] The volume of the receiving chamber is 10 mL to 15 mL; and / or,

[0044] The permeable area of ​​the animal skin is 0.5 cm² to 1.5 cm²; and / or,

[0045] The beauty serum is added via atomization at a rate of 0.2 mL / min to 1.5 mL / min; and / or,

[0046] The beauty serum is added by atomizing at a distance of 2 cm to 10 cm.

[0047] In some embodiments, the recipient fluid comprises 45% to 55% physiological saline and 45% to 55% ethanol by volume, the sum of which is 100%.

[0048] In some implementations, the following steps are also included:

[0049] Based on the concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the permeate, the permeation rate per unit time, cumulative permeation rate, steady-state permeation rate, linear range, and saturation point of each of the nicotinamide, tranexamic acid, and VC ethyl ether are obtained. Attached Figure Description

[0050] Figure 1 This is the MRM diagram of tranexamic acid in Example 1.

[0051] Figure 2 This is the calibration curve of tranexamic acid in Example 1.

[0052] Figure 3 This is the MRM diagram of nicotinamide in Example 1.

[0053] Figure 4 This is the calibration curve of nicotinamide in Example 1.

[0054] Figure 5 This is the MRM diagram of VC ethyl ether in Example 1.

[0055] Figure 6 The calibration curve of VC ethyl ether in Example 1.

[0056] Figure 7 This is a schematic diagram of the Franz diffusion cell in Example 1.

[0057] Explanation of reference numerals in the attached figures:

[0058] 100. Franz diffusion cell; 101. Supply chamber; 102. Receiving chamber; 103. Animal skin; 104. Stirring device; 200. Beauty solution; 300. Sampling needle. Detailed Implementation

[0059] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0061] Niacinamide, tranexamic acid, and vitamin C ethyl ether are widely used whitening and anti-inflammatory active ingredients in cosmetics, often added to creams, serums, lotions, and beauty mists, with high market demand. Niacinamide, tranexamic acid, and vitamin C ethyl ether are all highly polar, water-soluble small molecules. Traditional high-performance liquid chromatography (HPLC) methods are insufficient in separating these target functional components, resulting in low detection accuracy. Furthermore, to assess the permeability of these functional ingredients in practical applications, in vitro transdermal assays have become an important efficacy evaluation method. The commonly used experimental apparatus is the Franz diffusion cell; however, the concentration of the target component in the receiving solution of the Franz diffusion cell is usually very low, and it is often accompanied by a complex matrix background, such as skin metabolites, buffer residues, and formulation matrix components, making detection difficult. Ultraviolet detection methods also lack the sensitivity to meet the low-concentration detection requirements of transdermal assays. Therefore, improving the detection accuracy and sensitivity of simultaneously detecting multifunctional components has become an urgent technical challenge.

[0062] Based on this, one aspect of this application provides a method for detecting the components of a beauty serum, the beauty serum comprising niacinamide, tranexamic acid, and VC ethyl ether, the method comprising the following steps:

[0063] Add a protein precipitant to purify the beauty serum;

[0064] The purified beauty serum was analyzed by liquid chromatography-tandem mass spectrometry to obtain the chromatographic peak response values ​​of nicotinamide, tranexamic acid, and VC ethyl ether; among them...

[0065] The mass spectrometer employs alternating positive and negative ion ionization modes to perform multiple reaction monitoring on nicotinamide, tranexamic acid, and VC ethyl ether.

[0066] The concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the beauty serum were determined based on the chromatographic peak response values.

[0067] The above method targets beauty serums containing three active ingredients: nicotinamide, tranexamic acid, and VC ethyl ether. It simultaneously detects these three ingredients using liquid chromatography-tandem mass spectrometry (LC-MS / MS). This method removes matrix interferences from the beauty serum by adding a protein precipitant, reducing ion effects and chromatographic interference. In mass spectrometry, the method employs alternating positive and negative ion ionization modes. Multiple reaction monitoring (MRM) is performed on nicotinamide and tranexamic acid in positive ion ionization mode, and on VC ethyl ether in negative ion ionization mode. This achieves efficient separation and stable response of three compounds with different polarities and ionization characteristics—nicotinamide, VC ethyl ether, and tranexamic acid—within the same system, solving the problem of traditional methods being unable to accurately determine these three active ingredients simultaneously. Furthermore, this method lowers the detection limit, effectively meeting the detection needs when the concentration of active ingredients in the beauty serum is extremely low, and improving the detection accuracy for low-concentration, multi-target substances.

[0068] Understandably, alternating positive and negative ionization modes are an advanced ion source operating mode in liquid chromatography-mass spectrometry (LC-MS). This refers to the mass spectrometer rapidly and automatically switching the voltage polarity of the ionization source at different time intervals (usually milliseconds) during the same analysis, thereby causing compounds with different protonation difficulties to form either positive or negative ions, enabling simultaneous detection. For example, nicotinamide and tranexamic acid are detected in positive ionization mode, while VC ethyl ether is detected in negative ionization mode.

[0069] Furthermore, the mass concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the beauty serum were obtained based on the chromatographic peak response values.

[0070] In some implementations, multiple reaction monitoring includes the following steps:

[0071] Monitoring the transitions of nicotinamide from parent ion m / z 123 to daughter ion m / z 78 and / or from parent ion m / z 123 to daughter ion m / z 80; and / or,

[0072] For tranexamic acid, monitor the transitions from parent ion m / z 158 to daughter ion m / z 67 and / or the transitions from parent ion m / z 158 to daughter ion m / z 95; and / or; and / or,

[0073] For VC ethyl ether, monitor the transitions from the parent ion m / z 203 to the daughter ion m / z 85 and / or the transitions from the parent ion m / z 203 to the daughter ion m / z 131.

[0074] In some implementations, multiple reaction monitoring includes the following steps:

[0075] The transitions of the parent ion from m / z 123 to the daughter ion from m / z 78 and from m / z 123 to the daughter ion from m / z 80 were monitored for nicotinamide.

[0076] Transitions from parent ion m / z 158 to daughter ion m / z 67 and from parent ion m / z 158 to daughter ion m / z 95 were monitored for tranexamic acid.

[0077] The transitions of VC ethyl ether from the parent ion m / z 203 to the daughter ion m / z 85 and from the parent ion m / z 203 to the daughter ion m / z 131 were monitored. For nicotinamide, tranexamic acid, and VC ethyl ether, a dual screening method was used, detecting two ions to assess the transitions. This significantly reduced interference signals and clearly distinguished nicotinamide, VC ethyl ether, and tranexamic acid from the complex skin permeate, ensuring they did not interfere with each other and improving detection sensitivity and accuracy.

[0078] In some embodiments, the protein precipitant includes acetonitrile at a temperature of 0°C to 4°C.

[0079] In some of these embodiments, the operating conditions for liquid chromatography include:

[0080] The chromatographic column is a Waters BEH C18 column; and / or,

[0081] Column temperature is 35°C to 45°C; and / or,

[0082] The flow rate of the mobile phase is 0.3 mL / min to 0.6 mL / min.

[0083] In some embodiments, the operating conditions for liquid chromatography are as follows: the column is a Waters BEH C18 column; the column temperature is 35°C to 45°C; and the flow rate of the mobile phase is 0.3 mL / min to 0.6 mL / min.

[0084] In some embodiments, the mobile phase of the liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of acetic acid and mobile phase B is a methanol solution of acetic acid.

[0085] Understandably, gradient elution refers to the process in liquid chromatography where the mixing ratio of two or more solvents in the mobile phase is continuously changed according to a preset time gradient, thereby dynamically adjusting the elution capacity of the mobile phase to achieve efficient and rapid separation of components with different properties in complex mixtures.

[0086] Furthermore, the liquid chromatography employs gradient elution, which includes the following steps:

[0087] During the time period from 0 to t1, where t1 is 0 min to 1.0 min, the volume percentage of mobile phase A in the mobile phase is controlled to be 90% to 98%, and the volume percentage of mobile phase B in the mobile phase is controlled to be 2% to 10%.

[0088] During the time interval from t2 to t3, t2 is 1.0 min to 2.5 min and t3 is 2.5 min to 4.0 min. The volume percentage of mobile phase A in the mobile phase is controlled to be 5% to 15%, and the volume percentage of mobile phase B in the mobile phase is controlled to be 85% to 95%.

[0089] During the time interval from t4 to t5, t4 is 4.0 min ~ 4.1 min and t5 is 4.1 min ~ 7.0 min. The volume percentage of mobile phase A in the mobile phase is 90% ~ 98%, and the volume percentage of mobile phase B in the mobile phase is 2% ~ 10%.

[0090] In the gradient elution described above, a high-aqueous phase is used as the initial elution time from 0 to t1, allowing the highly polar tranexamic acid and nicotinamide to be moderately retained at the column head, preventing co-elution with the solvent peak. From t2 to t3, the organic phase is rapidly increased, intensifying the elution. The highly polar and weakly retained tranexamic acid and nicotinamide are eluted first, followed by the more strongly retained VC ethyl ether. From t4 to t5, the high-aqueous phase initial conditions are restored, providing a similar initial environment for the next injection. This gradient elution successfully separates nicotinamide, tranexamic acid, and VC ethyl ether of different polarities within 7 minutes, obtaining sharp, symmetrical chromatographic peaks. This provides excellent sample conditions for subsequent mass spectrometry analysis, improving the detection accuracy, analytical efficiency, and stability of the method described in this application.

[0091] In some implementations, the operating conditions for mass spectrometry include:

[0092] Waters H-Class LC coupled with TQS-micro triple quadrupole mass spectrometry; and / or,

[0093] The temperature of the ion source is 140°C to 180°C; and / or,

[0094] The ionization mode is an alternating ionization mode of ESI positive ions and ESI negative ions; and / or,

[0095] The solvent removal temperature is 400°C ~ 550°C; and / or,

[0096] The flow rate of the desolvation gas is 800 L / h ~ 1100 L / h; and / or,

[0097] The flow rate of the conical orifice gas is 1 L / h ~ 20 L / h.

[0098] Understandably, the alternating ionization mode of ESI positive and negative ions is an advanced ion source working mode in liquid chromatography-mass spectrometry. It refers to the mass spectrometer rapidly and automatically switching the voltage polarity of the electrospray ionization source at different time periods (usually milliseconds) during a single analysis, thereby causing compounds with different properties to form positive or negative ions respectively, so as to achieve simultaneous detection of them.

[0099] In some embodiments, the operating conditions of the mass spectrometer include: using a Waters H-Class LC coupled with a TQS-micro triple quadrupole mass spectrometer; an ion source temperature of 140°C to 180°C; an ionization mode of alternating ESI positive and ESI negative ion ionization; a desolvation temperature of 400°C to 550°C; a desolvation gas flow rate of 800 L / h to 1100 L / h; and a cone gas flow rate of 1 L / h to 20 L / h.

[0100] In some embodiments, the niacinamide content in the beauty serum is 0.0001% to 5% by mass, the tranexamic acid content is 0.0001% to 5% by mass, and the VC ethyl ether content is 0.0001% to 5% by mass.

[0101] In some embodiments, the beauty serum is selected from beauty atomizing liquid, beauty spray, toner, beauty essence, beauty gel, or beauty cream. The beauty serum simultaneously contains three active ingredients: niacinamide, tranexamic acid, and VC ethyl ether.

[0102] A second aspect of this application provides a method for testing the permeability of a beauty serum, the beauty serum comprising niacinamide, tranexamic acid, and VC ethyl ether, the method comprising the following steps:

[0103] An in vitro transdermal assay was conducted on the beauty serum, and the permeate was collected at different time points;

[0104] The concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the permeate were determined using the first method.

[0105] The above method targets beauty serums containing three active ingredients: nicotinamide, tranexamic acid, and VC ethyl ether. It simultaneously detects these three ingredients using liquid chromatography-tandem mass spectrometry (LC-MS / MS) under an in vitro transdermal assay model. This method removes complex matrix interferences such as buffer salts, ethanol, trace proteins, and skin metabolites from the permeate solution through purification with a protein-removing agent, reducing ion effects and chromatographic interference. The method employs alternating positive and negative ion ionization modes in mass spectrometry. Multiple reaction monitoring (MRM) is performed on nicotinamide and tranexamic acid in positive ion ionization mode and on VC ethyl ether in negative ion ionization mode. This achieves efficient separation and stable response of three compounds with different polarities and ionization characteristics—nicotinamide, VC ethyl ether, and tranexamic acid—within the same system, solving the problem of traditional methods' inability to accurately determine these three active ingredients simultaneously. Furthermore, this method lowers the detection limit, effectively meeting the detection requirements for extremely low permeate concentrations and improving the detection accuracy for low-concentration, multi-target substances.

[0106] The detection limit of the above method can be as low as 10 ng / mL.

[0107] In some embodiments, the method for testing the permeability of the beauty serum includes the following steps:

[0108] An in vitro transdermal assay was conducted on the beauty serum, and the permeate was collected at different time points;

[0109] The permeate was purified by adding a protein precipitant.

[0110] The purified permeate was analyzed by liquid chromatography-tandem mass spectrometry to obtain the chromatographic peak values ​​of nicotinamide, tranexamic acid, and VC ethyl ether; among them...

[0111] The mass spectrometer employs alternating positive and negative ion ionization modes to perform multiple reaction monitoring on nicotinamide, tranexamic acid, and VC ethyl ether.

[0112] The concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the permeate were obtained based on the chromatographic peak response values.

[0113] Understandably, the main difference between the above-mentioned method for detecting the permeability of beauty serums and the aforementioned method for detecting the components of beauty serums lies in the different objects of detection. In the method for detecting the components of beauty serums, the object of detection for liquid chromatography-tandem mass spectrometry is directly the purified beauty serum; in the method for detecting the permeability of beauty serums, the object of detection for liquid chromatography-tandem mass spectrometry is the purified permeate, which is the solution after the beauty serum has undergone an in vitro transdermal test.

[0114] In some embodiments, the method for testing the permeability of the beauty serum includes the following steps:

[0115] Ex vivo transdermal testing includes the following steps:

[0116] The excised animal skin was fixed between the supply chamber and the receiving chamber of the Franz diffusion cell, with the keratin layer of the animal skin facing the supply chamber and the dermis layer of the animal skin facing the receiving chamber.

[0117] Add the recipient fluid to the receiving chamber;

[0118] The beauty serum is added to the supply chamber and comes into contact with the stratum corneum of the animal skin to achieve transdermal penetration, and the permeate is obtained in the receiving chamber.

[0119] In some embodiments, the test apparatus for ex vivo transdermal testing includes a Franz diffusion cell and an atomizing device; the Franz diffusion cell includes a supply chamber, a receiving chamber, and an animal skin, with the animal skin located between the supply chamber and the receiving chamber, the stratum corneum of the animal skin facing the supply chamber and the dermis of the animal skin facing the receiving chamber, and the receiving chamber containing the receiving fluid; the atomizing liquid outlet of the atomizing device is connected to the supply chamber.

[0120] Furthermore, animal skins include, but are not limited to, pigskin.

[0121] Furthermore, the Franz diffusion cell is a vertical Franz diffusion cell.

[0122] In some embodiments, the recipient fluid comprises at least one of a mixture of physiological saline and ethanol, or a mixture of phosphate-buffered saline and ethanol.

[0123] In some embodiments, the temperature of the receptor fluid is 30°C to 37°C.

[0124] In some embodiments, the volume of the receiving chamber is 10 mL to 15 mL.

[0125] In some of these embodiments, the permeable area of ​​the animal skin is 0.5 cm² to 1.5 cm².

[0126] In some embodiments, the beauty serum is added via atomization at a rate of 0.2 mL / min to 1.5 mL / min. An atomizing device with a rate of 0.2 mL / min to 1.5 mL / min can be used for atomization.

[0127] In some embodiments, the beauty serum is atomized and added at a distance of 2 cm to 10 cm. The spray distance of the atomizing device is controlled to be 2 cm to 10 cm.

[0128] In some embodiments, the recipient fluid comprises 45% to 55% physiological saline and 45% to 55% ethanol by volume, the sum of which is 100%.

[0129] In some implementations, the following steps are also included:

[0130] Based on the concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the permeate, the permeate volume per unit time, cumulative permeate volume, steady-state permeate rate, linear range, and saturation point of nicotinamide, tranexamic acid, and VC ethyl ether were obtained.

[0131] In some embodiments, the purification treatment of the permeate includes the following steps:

[0132] Add a protein precipitant to the permeate to obtain a mixture;

[0133] The mixture is subjected to solid-liquid separation to remove protein precipitates and impurities, yielding a supernatant.

[0134] Filter the supernatant using a filter membrane.

[0135] The above purification process can effectively reduce proteins and impurities in complex permeate, providing high-quality samples for liquid chromatography-tandem mass spectrometry detection, thereby improving detection sensitivity and accuracy.

[0136] Furthermore, a solid-phase extraction purification step is included after membrane filtration.

[0137] Furthermore, the protein precipitant includes acetonitrile at temperatures between 0°C and 4°C. Acetonitrile disrupts the protein structure, causing denaturation and precipitation. The loss of the protein's native conformation exposes its internal hydrophobic regions, leading to intermolecular aggregation and further promoting the precipitation of interfering substances in the permeate. Additionally, some inorganic salts and impurities in the permeate, whose solubility in the acetonitrile solution is reduced, also co-precipitate with the protein, resulting in an unexpected purification effect. Low-temperature acetonitrile also helps inhibit the degradation of the target active ingredient, with a more pronounced inhibitory effect on the degradation of heat-sensitive VC ethyl ether. The low temperature also facilitates protein flocculation, forming a denser, more easily centrifuged precipitate, reducing the adsorption and encapsulation of the target active ingredient by the precipitate, further improving detection accuracy.

[0138] Furthermore, the filter membrane is an organic phase filter membrane with a pore size of 0.22μm~0.45μm.

[0139] In some embodiments, the purification treatment of the permeate includes the following steps:

[0140] Acetonitrile was added to the permeate as a precipitant to obtain a mixture;

[0141] The mixture is subjected to solid-liquid separation to remove protein precipitates and impurities, yielding a supernatant.

[0142] The supernatant was filtered through a filter membrane to obtain the filtrate;

[0143] The filtrate is purified by solid-phase extraction. This purification process, combining acetonitrile precipitation, membrane filtration, and solid-phase extraction, efficiently removes proteins and background impurities from the permeate, further improving detection sensitivity and accuracy, and is suitable for standardized laboratory procedures.

[0144] In some embodiments, solid-phase extraction purification is performed using a solid-phase extraction apparatus.

[0145] Furthermore, the solid phase extraction instrument uses a C18 SPE column.

[0146] To make the objectives, technical solutions, and advantages of this application clearer and more concise, the following specific embodiments are used for illustration, but this application is by no means limited to these embodiments. The embodiments described below are merely preferred embodiments of this application and can be used to describe this application, but should not be construed as limiting the scope of this application. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

[0147] To better illustrate this application, the following description, in conjunction with specific embodiments, further explains its content. The following are specific embodiments.

[0148] Example 1

[0149] The beauty serum and reagents used in Example 1 are as follows:

[0150] Beauty serum: A certain brand of revitalizing and repairing atomizing liquid (batch number: 20250601, referred to as atomizing liquid), the formula is: by mass percentage, including 5% niacinamide, 3% VC ethyl ether, 2% tranexamic acid, and the remainder is water, glycerin and ethanol and other excipients.

[0151] Ex vivo pig skin: Abdominal skin of healthy adult Large White pigs, with a thickness of 0.4 mm to 0.6 mm and an intact stratum corneum. Subcutaneous fat was removed, and the skin was rinsed with 0.9% physiological saline and then equilibrated in the saline for 30 minutes.

[0152] Receptor fluid: a freshly prepared mixture of physiological saline and ethanol in a 1:1 volume ratio.

[0153] Reagents: Standards of nicotinamide, VC ethyl ether and tranexamic acid (purity ≥98%, Sigma); acetonitrile (chromatographic grade, Merck); physiological saline (NaCl mass fraction 0.9%, Sinopharm Group); ultrapure water (prepared by Millipore ultrapure water system).

[0154] The instruments and equipment used in Example 1 are as follows:

[0155] Franz diffusion cell system: vertical Franz diffusion cell (acceptor volume 10 mL, permeation area 1.0 cm²), magnetic stirrer, constant temperature water bath;

[0156] Beauty atomizing device: a handheld atomizer for use with atomizing liquid (atomization volume 0.5 mL / min, spray distance 5 cm).

[0157] LC-MS / MS system: Waters H-Class LC coupled with TQS-micro triple quadrupole mass spectrometer;

[0158] Solid phase extraction apparatus: Supelco Visiprep™ DL solid phase extraction instrument (equipped with C18 SPE column, 500 mg / 6 mL).

[0159] Please refer to the test apparatus used in the ex vivo transdermal test in Example 1. Figure 7 The system includes a Franz diffusion cell 100 and an atomizing device (not shown). The Franz diffusion cell 100 includes a supply chamber 101, a receiving chamber 102, and an animal skin 103. The animal skin 103 is located between the supply chamber 101 and the receiving chamber 102, with the stratum corneum of the animal skin 103 facing the supply chamber 101 and the dermis of the animal skin 103 facing the receiving chamber 102. The receiving chamber 102 contains the receiving fluid, and the supply chamber 101 contains the beauty fluid 200. The atomizing fluid outlet of the atomizing device is connected to the supply chamber 101. The receiving chamber 102 is equipped with a stirring device 104 and a sampling port. The sampling port is used for sampling by a sampling needle 300. The stirring device 104 is a magnetic stirrer.

[0160] The test method for the permeation performance of the above-mentioned atomizing fluid is as follows:

[0161] S1: In vitro transdermal test:

[0162] Pre-treatment of excised pigskin: After shaving off the hair on the pigskin, cut a 0.5 mm thick skin piece with a scalpel and remove the subcutaneous fat. Rinse the skin piece three times with physiological saline and then place it in physiological saline at 32℃ for 30 minutes to ensure the integrity of the stratum corneum. The barrier function can be verified with an optical microscope and then set aside for later use.

[0163] Assemble the Franz diffusion cell system: Fix the excised pig skin between the supply chamber and the receiving chamber of the Franz diffusion cell, with the keratin layer of the excised pig skin facing upward towards the supply chamber and the dermis layer facing downward towards the receiving chamber. Add 10 mL of recipient fluid (a mixture of physiological saline and ethanol in a 1:1 volume ratio) to the receiving chamber. After removing air bubbles, stir the recipient fluid with a magnetic stir bar at the bottom of the receiving chamber at a stirring speed of 600 rpm. Maintain the temperature of the recipient fluid at 32±0.5°C by connecting a constant temperature water bath.

[0164] Nebulized drug delivery: Activate the cosmetic nebulizer and aim the nebulized liquid at the surface of the pig skin (keratin layer) at a spray distance of 5cm and a nebulization volume of 0.5 mL for 30 seconds. Immediately after the nebulization is completed, seal the supply chamber with sealing film and start timing (t=0).

[0165] Permeate samples were collected at different time points of 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h. 200 μL of permeate (i.e. the solution in the receiving chamber) was collected each time using a 1 mL syringe. 200 μL of fresh receiving fluid was added immediately after each sampling to maintain a constant volume. The permeate samples were refrigerated at 4°C and subsequent pretreatment was completed within 24 h.

[0166] The in vitro transdermal test was conducted in complete darkness to prevent oxidation of the active ingredients in the atomized liquid. The test was performed in a fume hood, and each group underwent three parallel tests.

[0167] S2: The collected permeate will be purified within 24 hours, specifically including the following steps:

[0168] Take 200 μL of the permeate sample and add 600 μL of acetonitrile (0℃~4℃) to precipitate proteins and impurities;

[0169] After vortexing, centrifuge at 4°C for 10 min to obtain the supernatant;

[0170] The supernatant was filtered through a filter membrane with a pore size of 0.22 μm to obtain the filtrate.

[0171] The filtrate was purified by solid-phase extraction: a C18 SPE column was activated with methanol and water in sequence, then the filtrate was loaded onto the column, followed by elution with methanol and collection of the target active ingredient eluent. The eluent was dried with nitrogen, and the residue obtained after drying was redissolved with the initial mobile phase (methanol and water, volume ratio B:A=5:95).

[0172] S3: Analyze the purified permeate using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0173] The operating conditions for liquid chromatography are as follows:

[0174] The chromatographic column used was a Waters BEH C18, 2.1 × 100 mm, 1.7 μm.

[0175] Column temperature: 40°C;

[0176] Flow rate of mobile phase: 0.3 mL / min;

[0177] Mobile phases: Phase A consists of acetic acid and water (acetic acid volume fraction is 0.1%), and Phase B consists of acetic acid and methanol (acetic acid volume fraction is 0.1%).

[0178] The gradient elution procedure is shown in Table 1 below:

[0179] Table 1

[0180]

[0181] The working conditions for mass spectrometry are as follows:

[0182] Instrumentation: Waters H-Class LC coupled with TQS-micro triple quadrupole mass spectrometer;

[0183] Ionization mode: ESI alternates between positive and negative;

[0184] Ion source temperature: 150°C, desolventization temperature: 450°C;

[0185] Gas flow rate: The flow rate of the desolvation gas is 900 L / h, and the flow rate of the conical gas is 2 L / h;

[0186] The multiple reaction monitoring (MRM) ion pair settings are shown in Table 2 below:

[0187] Table 2

[0188]

[0189] The data obtained from liquid chromatography-tandem mass spectrometry detection of nicotinamide, tranexamic acid and VC ethyl ether in the permeate in Example 1 were processed and analyzed.

[0190] 1) Standard curve and quantification

[0191] Prepare mixed standard solutions of three target active ingredients (concentration gradient of 10 ng / mL to 1000 ng / mL). After purifying the mixed standard solutions according to step S2 above, perform LC-MS / MS analysis. Plot a standard curve with the concentration of the target active ingredient on the x-axis and the peak area ratio (target active ingredient / internal standard) on the y-axis. The linear equation and correlation coefficient are as follows:

[0192] Tranexamic acid: y = 3091.72x + 5269.4, R 2 =0.999338;

[0193] Nicotinamide: y = 214.963x + 566.009, R 2 =0.999368;

[0194] VC ethyl ether: y = 8.57221x + 13.8701, R 2 =0.999288;

[0195] in, Figure 1The MRM diagram of tranexamic acid. Figure 2 This is the calibration curve for tranexamic acid. Figure 3 The MRM diagram for nicotinamide is shown below. Figure 4 This is the calibration curve for nicotinamide. Figure 5 The MRM diagram for VC ethyl ether is shown. Figure 6 This is a calibration curve for VC ethyl ether; the horizontal axis of the calibration curve represents the concentration of the target active ingredient, and the vertical axis represents the peak area of ​​the target active ingredient.

[0196] Figure 1 , Figure 3 and Figure 5 The mid-spectral peaks are clear, sharp, and well separated, R 2 A value >0.995 indicates good linearity of the calibration curve, demonstrating that after the aforementioned purification process, LC-MS / MS can effectively separate and qualitatively and quantitatively analyze the three active ingredients: tranexamic acid, nicotinamide, and VC ethyl ether.

[0197] 2) Results of concentration determination of target active ingredient in permeate

[0198] The mass concentrations of the target active ingredient in the permeate at each time point, determined by LC-MS / MS (three parallel experiments per group, mean ± SD), are shown in Table 3 below:

[0199] Table 3

[0200]

[0201] 3) Calculation of various transdermal permeability parameters

[0202] ① Osmotic rate per unit time (Qi, μg / cm²)

[0203] Definition of permeation per unit time: The amount of component permeated through the skin within adjacent sampling time points (t(i-1)~t(i)), reflecting the permeation efficiency during that time period;

[0204] Formula for calculating permeability per unit time (corrected volume method): ,

[0205] Ci: Concentration of the target component in the permeate at the i-th time point (μg / mL, determined by LC-MS / MS);

[0206] V: Acceptance cell volume (fixed, e.g., 10 mL);

[0207] Vs: Sampling volume per sample (fixed, 200 μL = 0.2 mL);

[0208] A: Transmittance area (1.0 cm²);

[0209] : The total amount of components removed in the first i-1 samplings (because Vs is replenished after each sampling, the amount lost in the previous sampling needs to be corrected).

[0210] ② Cumulative permeability (Qcum(t), μg / cm²)

[0211] Definition of cumulative penetration: The total amount of penetration from the start of the experiment (t=0) to a certain time point t, reflecting the total amount of ingredients that pass through the skin.

[0212] The method for calculating cumulative permeability is the sum of the permeability per unit time at each time point:

[0213]

[0214] Where n is the number of samplings from t=0 to t (e.g., t=2h corresponds to n=3, which is the sum of Qi at the three time points of 0.5h, 1h, and 2h).

[0215] ③ Permeability curve

[0216] Definition of permeation curve: A curve (Qcum-t curve) plotted with time t (h) as the abscissa and cumulative permeation amount Qcum (t) (μg / cm²) as the ordinate, which intuitively reflects the dynamic process of transdermal permeation of components.

[0217] ④ Permeation rate (Jss, μg / (cm²·h), steady-state permeation rate)

[0218] Definition of penetration rate: After the penetration curve enters the linear stage (steady state), the rate of change of the cumulative penetration amount over time reflects the stable efficiency of the ingredients through the skin (core parameter).

[0219] The method for calculating the permeation rate is as follows: First, determine the linear interval, then perform piecewise linear regression on the Qcum-t curve, and select the correlation coefficient R. 2 The continuous time interval ≥0.98 is taken as the linear interval (steady-state stage); then the slope is calculated, and within the linear interval, Qcum(t) and t are linearly fitted (Qcum=Jss×t+b), and the slope Jss is the permeation rate.

[0220] ⑤ Linear interval (t_start~t_end, h)

[0221] Definition of linear interval: In the permeation curve, Qcum(t) has a linear relationship with t (R0). 2 The time period (≥0.98) reflects the duration of stable diffusion of the ingredient through the skin.

[0222] Method for determining linear intervals: Perform linear regression on Qcum-t data for different time periods and calculate Rm. 2 Select R2 The largest and closest continuous interval to 1 (e.g., t=2~8h).

[0223] ⑥ Saturation point (ts, h; Qss, μg / cm²)

[0224] The definition of saturation point: After the linear interval ends, the point at which Qcum(t) tends to flatten out with time (the slope decreases significantly) and the corresponding cumulative penetration amount (Qss) reflects the upper limit of the skin's ability to penetrate ingredients.

[0225] The saturation point is determined as follows: when the instantaneous slope (ΔQcum / Δt) after the linear interval drops to below 50% of the steady-state permeation rate Jss, the corresponding t is ts, and Qcum(ts) is Qss.

[0226] Based on the mass concentration data in Table 3, the cumulative permeability (Qcum), steady-state permeability (Jss), linear interval, and saturation point were calculated, and the results are shown in Table 4 below:

[0227] Table 4

[0228]

[0229] As shown in Table 3 above, in Example 1, the purification process removed complex background substances such as buffer salts, ethanol, trace proteins, and skin metabolites from the permeate, reducing ion effects and chromatographic interference. At the same time, by using gradient elution and alternating positive and negative ion ionization modes in liquid chromatography, efficient separation and stable response of three compounds with different polarities and ionization characteristics—nicotinamide, VC ethyl ether, and tranexamic acid—were achieved in the same system, improving the detection accuracy of low-concentration, multi-target substances.

[0230] Furthermore, as can be seen from Table 3 above, the mass concentration of nicotinamide in the permeate collected at the same time was greater than that of VC ethyl ether and tranexamic acid, indicating that nicotinamide had the best transdermal permeation effect in this nebulizer, followed by VC ethyl ether, and tranexamic acid had the worst effect.

[0231] As shown in Table 4 above, the steady-state permeation rate of nicotinamide in the permeate is 9.8 μg / (cm²). 2 The cumulative penetration rate over 24 hours was 108.0 μg / cm², and the saturation point was 99.0 μg / cm², both significantly higher than that of VC ethyl ether and tranexamic acid (P<0.05, ANOVA). This further demonstrates that nicotinamide has the best transdermal penetration effect in this atomizing fluid, followed by VC ethyl ether, and tranexamic acid has the worst effect. It also indicates that nicotinamide is more suitable as the core transdermal active ingredient of this type of atomizing fluid.

[0232] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0233] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for detecting the components of a beauty serum, characterized in that, The beauty serum comprises niacinamide, tranexamic acid, and vitamin C ethyl ether, and the method includes the following steps: The beauty essence is purified by adding a protein precipitant; The purified beauty serum was subjected to liquid chromatography-tandem mass spectrometry (LC-MS / MS) to obtain the chromatographic peak response values ​​of nicotinamide, tranexamic acid, and VC ethyl ether; wherein... The nicotinamide, tranexamic acid, and VC ethyl ether were subjected to multiple reaction monitoring in the mass spectrometer using alternating positive and negative ion ionization modes. The concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the beauty serum were determined based on the chromatographic peak response values.

2. The method for detecting the components of a beauty serum as described in claim 1, characterized in that, The multiple reaction monitoring includes the following steps: Monitoring the transitions of nicotinamide from parent ion m / z 123 to daughter ion m / z 78 and / or from parent ion m / z 123 to daughter ion m / z 80; and / or, For tranexamic acid, monitor the transitions from parent ion m / z 158 to daughter ion m / z 67 and / or the transitions from parent ion m / z 158 to daughter ion m / z 95; and / or; and / or, For VC ethyl ether, monitor the transitions from the parent ion m / z 203 to the daughter ion m / z 85 and / or the transitions from the parent ion m / z 203 to the daughter ion m / z 131.

3. The method for detecting the components of a beauty serum as described in claim 1, characterized in that, The protein precipitant comprises acetonitrile at a temperature of 0°C to 4°C.

4. The method for detecting the components of a beauty serum as described in any one of claims 1 to 3, characterized in that, The operating conditions for the liquid chromatography include: The chromatographic column is a Waters BEH C18 column; and / or, Column temperature is 35°C to 45°C; and / or, The flow rate of the mobile phase is 0.3 mL / min to 0.6 mL / min.

5. The method for detecting the components of a beauty serum as described in any one of claims 1 to 3, characterized in that, The mobile phase of the liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of acetic acid and mobile phase B is a methanol solution of acetic acid. Optionally, the liquid chromatography performs gradient elution, which includes the following steps: During the time period from 0 to t1, where t1 is 0 min to 1.0 min, the volume percentage of mobile phase A in the mobile phase is controlled to be 90% to 98%, and the volume percentage of mobile phase B in the mobile phase is controlled to be 2% to 10%. During the time interval from t2 to t3, t2 is 1.0 min to 2.5 min and t3 is 2.5 min to 4.0 min. The volume percentage of mobile phase A in the mobile phase is controlled to be 5% to 15%, and the volume percentage of mobile phase B in the mobile phase is controlled to be 85% to 95%. During the time interval from t4 to t5, t4 is 4.0 min ~ 4.1 min and t5 is 4.1 min ~ 7.0 min. The volume percentage of mobile phase A in the mobile phase is 90% ~ 98%, and the volume percentage of mobile phase B in the mobile phase is 2% ~ 10%.

6. The method for detecting the components of a beauty serum as described in any one of claims 1 to 3, characterized in that, The operating conditions for the mass spectrometer include: Waters H-Class LC coupled with TQS-micro triple quadrupole mass spectrometry; and / or, The temperature of the ion source is 140°C to 180°C; and / or, The ionization mode is an alternating ionization mode of ESI positive ions and ESI negative ions; and / or, The solvent removal temperature is 400°C ~ 550°C; and / or, The flow rate of the desolvation gas is 800 L / h ~ 1100 L / h; and / or, The flow rate of the conical orifice gas is 1 L / h ~ 20 L / h.

7. The method for detecting the components of a beauty serum as described in any one of claims 1 to 3, characterized in that, By mass percentage, the beauty serum contains 0.0001% to 5% niacinamide, 0.0001% to 5% tranexamic acid, and 0.0001% to 5% VC ethyl ether; and / or, The beauty essence is selected from beauty atomizing liquid, beauty spray, toner, beauty serum, beauty gel or beauty cream.

8. A method for testing the permeability of a beauty serum, characterized in that, The beauty serum comprises niacinamide, tranexamic acid, and vitamin C ethyl ether, and the method includes the following steps: An in vitro transdermal test was conducted on the beauty serum, and the permeate was collected at different time points; The concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the permeate are determined by the method described in any one of claims 1 to 7.

9. The method for testing the permeability of a beauty serum as described in claim 8, characterized in that, Includes the following steps: The ex vivo transdermal test includes the following steps: The excised animal skin is fixed between the supply chamber and the receiving chamber of the Franz diffusion cell, with the keratin layer of the animal skin facing the supply chamber and the dermis layer of the animal skin facing the receiving chamber. Add the recipient fluid to the receiving chamber; The beauty solution is added to the supply chamber and comes into contact with the stratum corneum of the animal skin to achieve transdermal penetration, and the permeated solution is obtained in the receiving chamber.

10. The method for testing the permeability of a beauty serum as described in claim 9, characterized in that, The recipient fluid comprises at least one of the following: a mixture of physiological saline and ethanol, or a mixture of phosphate-buffered saline and ethanol; and / or, The temperature of the receptor solution is 30°C to 37°C; and / or, The volume of the receiving chamber is 10 mL to 15 mL; and / or, The permeable area of ​​the animal skin is 0.5 cm² to 1.5 cm²; and / or, The beauty serum is added via atomization at a rate of 0.2 mL / min to 1.5 mL / min; and / or, The beauty serum is added by atomizing at a distance of 2 cm to 10 cm.

11. The method for testing the permeability of a beauty serum as described in claim 10, characterized in that, The receptor fluid comprises 45% to 55% physiological saline and 45% to 55% ethanol by volume, with the sum of the two being 100%.

12. The method for testing the permeability of a beauty serum as described in any one of claims 8 to 11, characterized in that, It also includes the following steps: Based on the concentrations of nicotinamide, tranexamic acid, and VC ethyl ether in the permeate, the permeation rate per unit time, cumulative permeation rate, steady-state permeation rate, linear range, and saturation point of each of the nicotinamide, tranexamic acid, and VC ethyl ether are obtained.

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