Method for determining the content of ectoine in cosmetics by cation exchange

Abstract

The application discloses a method for determining the content of ectoine in cosmetics by a cation exchange method. The method comprises the following steps: using ectoine as a standard sample, dissolving the ectoine standard sample with a mobile phase to form a standard solution, analyzing by a cation exchange-non-suppressed ion chromatography conductivity detection device, and establishing a standard working curve of ectoine; using the mobile phase and n-hexane to extract and purify the cosmetic to be tested to obtain a cosmetic sample solution to be tested; analyzing the cosmetic sample solution to be tested by the cation exchange-non-suppressed ion chromatography conductivity detection device, and comparing with the standard working curve, so as to determine the content of ectoine in the cosmetic. The method can effectively extract ectoine in different substrates by selecting a suitable extraction solvent and a convenient pretreatment operation for various complex cosmetic substrates, and can effectively separate and detect ectoine in the cosmetic and effectively eliminate interference by using the cation exchange-non-suppressed ion chromatography conductivity detection device.

Description

Technical Field

[0001] This invention belongs to the field of testing technology, specifically relating to a method for determining the ectoine content in cosmetics using a cation exchange method. Background Technology

[0002] Ectoin (EC), also known as tetrahydropyrimidine or tetrahydromethylpyrimidine carboxylic acid, with the chemical name 2-methyl-1,4,5,6,-tetrahydropyrimidine-4-carboxylic acid, is a cyclic amino acid derivative. First discovered in 1985 by Professor Galinski in the Egyptian desert, who found that desert halophiles, under conditions of high temperature, dryness, strong UV radiation, and high salinity, produce a natural protective component on their outer cell layer—ectoin (EC). Ectoin (EC) not only balances cell osmotic pressure but also provides excellent protection for enzymes, DNA, proteins, nucleic acids, cell membranes, and the entire cell under adverse conditions such as high temperature, extreme cold, drought, extreme pH, high pressure, high salinity, and high radiation. Studies have shown that ectoin (EC) has the effects of isolating irritants, enhancing skin immunity, accelerating cell repair, repairing UV damage, restoring skin's water-locking ability, and anti-aging and anti-wrinkle effects. It is currently widely used in enzyme preparations, biomedicine and skin care products and other related fields.

[0003] Due to its various skin-repairing and protective effects, ectoine is increasingly widely used in cosmetics. However, current detection of ectoine mainly employs high-performance liquid chromatography (HPLC), primarily focusing on the detection of products from halophilic bacteria. Weng Nanhai et al. used HPLC to detect the tetrahydropyrimidine-producing ability of a salt-tolerant denitrifying bacterium. They used a modified ethanol extraction method to extract intracellular tetrahydropyrimidine. The chromatographic conditions included an SBC18 column (4.6 mm × 250 mm, 5 μm), a mobile phase of water / acetonitrile (V:V, 10:90), a flow rate of 1.0 mL / min, a column temperature of 30℃, an injection volume of 2 μL, and detection of ectoine at a wavelength of 204 nm with a retention time of 4 min. Li Yaodong et al. extracted tetrahydropyrimidine, a compatible substance accumulated intracellularly in moderately halophilic bacteria from Qinghai Lake, using 80% ethanol. They then qualitatively identified the presence of intracellular tetrahydropyrimidine using TLC and detected it using high-performance liquid chromatography (HPLC). The mobile phase was acetonitrile / methanol (V:V, 95:5), the column was an EClipseXDB-C18 (150×4.6 mm), the column temperature was 20℃, the flow rate was 1.0 mL / min, and the detection wavelength was 204 nm. Under these chromatographic conditions, the retention time of ectoine was 1.400–1.429 min. However, due to the accumulation of hydroxytetrahydropyrimidine, which has similar chemical properties to tetrahydropyrimidine, in the bacterial cells, the HPLC method could not separate the two, resulting in some strains exhibiting double peaks or shoulder peaks in their chromatograms. Li Haiyu et al. effectively separated tetrahydropyrimidine and its derivative hydroxytetrahydropyrimidine, a compatible substance in moderately halophilic bacteria, using ultra-high performance liquid chromatography (UPLC). This reduced interference between the detected substances, and UPLC only required a 1 μL sample dose for analysis. However, the equipment is expensive and its widespread use is limited by the current research environment. Riis V et al. used an anion exchange-pulse ampoule detection method to detect ectoin and other compatible substances, which can be used for strain screening. This method involves alkaline hydrolysis of ectoin followed by detection of its hydrolysis product, γ-N-acetyldiaminobutyric acid (γ-N-acetyldiaminobutyric acid). However, the elution procedure is complex and cannot directly detect the ectoin content. Tang Yuping et al. determined the content of ectoine in cosmetics by high performance liquid chromatography. They used an Agilent Poroshell 120 EC-C 18 column (100 mm × 3.0 mm, 2.7 μm) for separation, with methanol and phosphate system as the mobile phase, flow rate of 0.8 mL / min, column temperature of 30 ℃, and detection wavelength of 210 nm to separate ectoine in cosmetics.

[0004] The main problem with the above liquid chromatography method for analyzing ectoine is that the detection wavelength is between 204 nm and 210 nm, which is close to the vacuum ultraviolet region. Absorption interference is significant at the end of this wavelength range. Although the standard solution of ectoine shows good peak shape in liquid chromatography, it is prone to interference when analyzing actual cosmetic samples, especially complex matrices such as creams and lotions (see...). Figure 3 Meanwhile, ectoine has strong hydrophilic properties, resulting in short retention times and poor separation on conventional C18 chromatographic columns, where the main peak cannot be effectively separated from interfering peaks. Summary of the Invention

[0005] The main objective of this invention is to provide a method for determining the ectoine content in cosmetics using a cation exchange method, thereby overcoming the shortcomings of the prior art.

[0006] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0007] This invention provides a method for determining the ectoine content in cosmetics using a cation exchange method, comprising:

[0008] Ectocin was used as a standard. The ectocin standard was dissolved in the mobile phase to form a standard solution. The solution was analyzed by a cation exchange-non-inhibited ion chromatography conductivity detection device to establish a standard working curve for ectocin.

[0009] The cosmetic sample was extracted and purified using a mobile phase and n-hexane to obtain the cosmetic sample solution.

[0010] Furthermore, the cosmetic test solution was analyzed using a cation exchange-non-inhibited ion chromatography conductivity detection device, and compared with the standard working curve to determine the content of ectoine in the cosmetic.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: For cosmetics containing various complex matrices such as oils, powders, creams, emulsions, and surfactants, the present invention effectively extracts ectoine by selecting a suitable extraction solvent under certain pH conditions. After eliminating the influence of matrices such as oils, cation exchange-non-inhibitory ion chromatography conductivity detection is used, which can effectively detect ectoine in various cosmetics. The sensitivity is moderate, the separation of ectoine is good, the retention time on the ion exchange column is long, the influence of the matrix is ​​small, and interference can be effectively eliminated. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is a standard curve diagram of ectoine in a typical embodiment of the present invention;

[0014] Figure 2 This is an ion chromatogram of a standard solution of ectoine in a typical embodiment of the present invention;

[0015] Figure 3 The method involves using liquid chromatography to analyze the interference of ectoine in actual cosmetic samples. Detailed Implementation

[0016] In view of the deficiencies of the prior art, the inventors of this case, through long-term research and extensive practice, have proposed the technical solution of this invention. The technical solution of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0017] Specifically, as one aspect of the technical solution of this invention, a method for determining the ectoine content in cosmetics using a cation exchange method includes:

[0018] Ectocin was used as a standard. The ectocin standard was dissolved in the mobile phase to form a standard solution. The solution was analyzed by a cation exchange-non-inhibited ion chromatography conductivity detection device to establish a standard working curve for ectocin.

[0019] The cosmetic sample was extracted using a mobile phase and n-hexane to obtain the cosmetic sample solution.

[0020] Furthermore, the cosmetic test solution was analyzed using a cation exchange-non-inhibited ion chromatography conductivity detection device, and compared with the standard working curve to determine the content of ectoine in the cosmetic.

[0021] In some more specific embodiments, the method specifically includes: using ectoine standards, dissolving and mixing ectoine with the mobile phase to prepare a series of standard solutions with different ectoine concentrations, and under certain pH conditions, exchanging H+ in the mobile phase with ectoine cations. +Ions were analyzed using a non-suppressed ion chromatography conductivity detection device, and a standard working curve of peak area versus concentration for ectoine was established; wherein, the standard working curve is y = 0.0264x + 0.0192, R 2 =0.9999.

[0022] Furthermore, the concentration of ectoine in the standard solution is 5–500 mg / L.

[0023] In some more specific embodiments, the mobile phase includes methanesulfonic acid and acetonitrile.

[0024] Furthermore, the concentration of methanesulfonic acid in the mobile phase is 5.0-6.0 mmol / L.

[0025] Furthermore, the acetonitrile content in the mobile phase is 15-25 wt%, and the pH value is 1.8-2.3.

[0026] In some more specific implementation schemes, the method specifically includes: mixing and dispersing the mobile phase, n-hexane and the cosmetic to be tested to form a mixed dispersion, and then purifying it by ultrasonication, centrifugation, filtration and layering to obtain the cosmetic test solution.

[0027] Furthermore, the mass-to-volume ratio of the mixed dispersion, n-hexane, and the cosmetic product to be tested is 50 mL: 2 mL: 0.5 g.

[0028] Furthermore, the centrifugation process is carried out at a speed of 10,000 r / min and a centrifugation time of 5 to 10 min.

[0029] Furthermore, the method includes: filtering the supernatant obtained from centrifugation using a filter membrane with a pore size of 0.22 μm.

[0030] In some more specific implementations, the parameters used in the cation exchange-non-suppressed ion chromatography conductivity detection device include: a PRP-X200 Cation Exchange column; a mobile phase of 5.4 mmol / L methanesulfonic acid-15% acetonitrile; a flow rate of 2.0 mL / min; a column temperature of 25 °C; and an injection volume of 25 μL.

[0031] In some more specific implementation schemes, when the spiking recovery test is performed on the cosmetic test solution, the average recovery rate of ectoine is 99.6% to 123.77%, and the precision is ≤6.35%.

[0032] In some more specific implementation schemes, the detection limit of ectoine in cosmetics based on the ion exchange method is 0.6 mg / L.

[0033] In some more specific implementation schemes, the cosmetic to be tested includes creams, lotions, or liquids.

[0034] In some more specific embodiments, the method for determining the ectoine content in cosmetics based on ion exchange includes:

[0035] (1) Sample pretreatment:

[0036] Weigh 0.5g of sample into a 50mL colorimetric tube, add an appropriate amount of mobile phase and 2mL of n-hexane, vortex to disperse, and bring the volume to 50mL with the mobile phase. After sonicating for 15min, centrifuge the extract at 10000r / min for 5min, remove the supernatant and filter it through a 0.22μm filter membrane. The filtrate is ready for testing.

[0037] (2) Prepare the standard working curve

[0038] Weigh 40 mg of ectoine standard (accurate to 0.01 mg), dissolve it in an appropriate amount of mobile phase and dilute to 20 mL to prepare a 2 mg / mL standard stock solution. Store refrigerated and protected from light. Shelf life is 6 months. Take different volumes of the standard stock solution and dilute with mobile phase to prepare a series of ectoine standard solutions with mass concentrations of 5, 10, 20, 50, 100, and 200 mg / L.

[0039] The ion chromatography conditions for this method are as follows:

[0040] Chromatographic column: PRP-X200 Cation Exchange (250mm×4.6mm×5m) cation exchange column;

[0041] Mobile phase: 5.4 mmol / L methanesulfonic acid - 15% acetonitrile;

[0042] Flow rate: 2.0 mL / min;

[0043] Column temperature: 25℃;

[0044] Injection volume: 25 μL.

[0045] The standard solution showed good linearity in the concentration range of 5–500 mg / L. The limit of detection (LOD) of ectoine in cosmetics was 0.603 mg / L, and the limit of quantification (LOD) was 2.412 mg / L. Spiking recovery tests were conducted, and the average recoveries at three spiking levels (500 μg, 2000 μg, and 4000 μg, n=6) were 99.6%–123.77%, with a precision ≤6.35%.

[0046] This invention aims to establish a method for detecting ectoine content in cosmetics using ion exchange. The method employs a cation exchange-non-suppressed ion chromatography conductivity detector. The separation mechanism is primarily ion exchange, based on the reversible exchange between dissociable ions on the ion exchange resin and solute ions with the same charge in the mobile phase. During the ion exchange process, the mobile phase continuously provides ions with the same charge as the equilibrium ions at the ion exchange sites in the stationary phase. These equilibrium ions, H... + (The eluent ions in the eluent) combine with the opposite charges at the ion exchange sites of the stationary phase and maintain charge balance. After injection, the amino groups in the ectoine molecule react with H under acidic conditions. + Combine to form positively charged ectoine H + Cations and leaching ions H + Competition for charge sites on the stationary phase leads to the temporary retention of ectoine cations by the stationary phase. Simultaneously, the retained ectoine cations are further attacked by the eluent ions H+ in the eluent. + The ectoine cation is displaced and eluted from the column. When the ectoine cation passes through the detector, it affects the conductivity of the detector, thereby generating an electrical signal, which in turn detects the ectoine in the sample. This invention effectively extracts ectoine and eliminates interference in cosmetics containing various complex matrices such as oils, powders, and surfactants under certain pH conditions, with a suitable mobile phase and certain pretreatment operations. It uses a non-suppressed conductivity detector mode for detection, which can effectively separate and detect ectoine in cosmetics. The operation is simple, the results are accurate, and interference is effectively eliminated.

[0047] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides detailed implementation methods and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0048] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0049] Ectoin (EC), also known as tetrahydromethylpyrimidine carboxylic acid, has a historical usage of 7% in leave-on cosmetics listed in the "Catalogue of Used Cosmetic Ingredients (2021 Edition)". According to an assessment report by the Australian National Institute of Industrial Chemicals Registration and Evaluation (NICNAS) (now AICIS), the risk of this ingredient is within an acceptable range when its concentration in cosmetics does not exceed 3%.

[0050] The steps for establishing a standard operating curve in the following embodiments are as follows:

[0051] Weigh 40 mg of ectoine standard (accurate to 0.01 mg), dissolve it in an appropriate amount of mobile phase and dilute to 20 mL to prepare a 2 mg / mL standard stock solution. Store refrigerated and protected from light. Shelf life is 6 months. Take different volumes of the standard stock solution and dilute with mobile phase to prepare a series of ectoine standard solutions with mass concentrations of 5, 10, 20, 50, 100, and 200 mg / L.

[0052] The ion chromatography conditions for this method are as follows:

[0053] Chromatographic column: PRP-X200 Cation Exchange (250mm×4.6mm×5m) cation exchange column;

[0054] Mobile phase: 5.4 mmol / L methanesulfonic acid - 15% acetonitrile;

[0055] Flow rate: 2.0 mL / min;

[0056] Column temperature: 25℃;

[0057] Injection volume: 25 μL.

[0058] The standard curve of ectoine is shown below. Figure 1 As shown, the standard working curve is: y = 0.0264x + 0.0192, R0 2 =0.9999 (the peak area on the ordinate represents the integral value of conductivity and retention time); the standard solution concentrations of ectoine are 5.9, 11.81, 29.52, 59.05, 118.09, and 236.18 μg / mL, and the ion chromatograms are as follows. Figure 2 As shown, the standard liquid chromatogram of ectoine is as follows: Figure 3 As shown, interference can be observed in liquid chromatography.

[0059] Example 1

[0060] Seven different cosmetics were selected (Ectoin Fermented Repair Essence Water, Ectoin Fermented Repair Essence Lotion (Refreshing Type), Ectoin Fermented Repair Essence Lotion (Moisturizing Type), Ectoin Baby Soothing Essence, White Olive Ectoin Moisturizing and Soothing Essence Body Lotion, White Fig Ectoin Multi-Effect Moisturizing and Repairing Essence Cream, and Ectoin Moisturizing Body Lotion, and named Sample 1 to Sample 7 respectively). 0.5g of each sample was weighed into a 50mL colorimetric tube, an appropriate amount of mobile phase and 2ml of n-hexane were added, the mixture was vortexed, and the volume was adjusted to 50mL with the mobile phase. After sonication for 15min, the extract was centrifuged at 10000r / min for 5min. The supernatant was collected and filtered through a 0.22μm filter membrane. The filtrate was then tested to obtain the cosmetic test solution.

[0061] The cosmetic test solution was analyzed using a cation exchange-non-inhibited ion chromatography conductivity detector, and compared with the aforementioned standard working curve to obtain the content of ectoine in the cosmetic, as shown in Table 1.

[0062] Table 1. Ectoin content in samples

[0063] Ikdoin 0.198％ 1.195％ 0.125％ 0.095％ 0.110％ 0.121％ 0.086％

[0064] Example 2

[0065] A cosmetic blank matrix (emulsion) was selected, and three spiking levels of 500 μg, 2000 μg, and 4000 μg were set. Six replicates were made for each spiking level. The spiking test results are shown in Table 2.

[0066] Table 2 Results of the spiked test

[0067]

[0068]

[0069] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0070] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. A method for determining the ectoine content in cosmetics using cation exchange chromatography, characterized in that, include: Ectocin was used as a standard. The ectocin standard was dissolved in the mobile phase to form a standard solution. The solution was analyzed by a cation exchange-non-inhibited ion chromatography conductivity detection device to establish a standard working curve for ectocin. The cosmetic sample was extracted and purified using a mobile phase and n-hexane to obtain the cosmetic sample solution. Furthermore, the cosmetic test solution was analyzed using a cation exchange-non-inhibited ion chromatography conductivity detection device, and compared with the standard working curve to determine the content of ectoine in the cosmetic. The mobile phase comprises methanesulfonic acid and acetonitrile; the concentration of methanesulfonic acid in the mobile phase is 5.0-6.0 mmol / L; the content of acetonitrile in the mobile phase is 15-25 wt%, and the pH value is 1.8-2.

3.

2. The method according to claim 1, characterized in that, Specifically, it includes: Ectocin was used as a standard. A series of standard solutions with different ectocin concentrations were prepared by dissolving and mixing ectocin with the mobile phase. These solutions were analyzed using a cation exchange-non-suppressed ion chromatography conductivity detection device to establish a standard working curve for the peak area versus concentration of ectocin. The standard working curve was defined as y = 0.0264x + 0.0192, R0. 2 =0.9999.

3. The method according to claim 2, characterized in that: The concentration of ectoine in the standard solution is 5~500 mg / L.

4. The method according to claim 1, characterized in that, Specifically, it includes: Methanesulfonic acid, acetonitrile, and n-hexane are mixed and dispersed with the cosmetic to be tested to form a mixed dispersion, which is then subjected to ultrasonication, centrifugation, and filtration to obtain the cosmetic test solution.

5. The method according to claim 4, characterized in that: The mass-to-volume ratio of the mixed dispersion, n-hexane, and the cosmetic product to be tested was 50 mL: 2 mL: 0.5 g; And / or, the centrifugation process is carried out at a speed of 10,000 r / min and a centrifugation time of 5 to 10 min; And / or, the method includes: filtering the supernatant obtained from centrifugation using a filter membrane with a pore size of 0.22 μm.

6. The method according to claim 1, characterized in that, The parameters used in the cation exchange-non-suppressed ion chromatography conductivity detection device include: a PRP-X200 cation exchange column; a mobile phase of 5.4 mmol / L methanesulfonic acid-15% acetonitrile; a flow rate of 2.0 mL / min; a column temperature of 25℃; and an injection volume of 25 μL.

7. The method according to claim 1, characterized in that: When the spiking recovery test was performed on the cosmetic test solution, the average recovery rate of ectoine was 99.6%~123.77%, and the precision was ≤6.35%.

8. The method according to claim 1, characterized in that: The detection limit for ectoine in cosmetics determined by ion exchange method is 0.6 mg / L.

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