CN114324219A - Method for detecting content of retinol and 6 retinol derivatives in cosmetics - Google Patents
Method for detecting content of retinol and 6 retinol derivatives in cosmetics Download PDFInfo
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Abstract
The invention provides a method for detecting the content of retinol and 6 retinol derivatives in cosmetics, which comprises the following steps: adding a cosmetic sample into a first solvent for dispersion, adding a second solvent for ultrasonic extraction, filtering, collecting a sample solution obtained from the filtrate, determining the detection wavelength by ultraviolet absorption spectrometry, and performing qualitative and quantitative detection by high performance liquid chromatography to determine the content of retinol and 6 retinol derivatives in the sample solution. The method for detecting the content of the retinol and the 6 retinol derivatives in the cosmetics has the characteristics of simple and convenient pretreatment, reliable sample separation, accurate quantitative determination and the like, and has the advantages of good detection recovery rate, high accuracy, high sensitivity and low detection limit.
Description
Technical Field
The invention belongs to the technical field of detection of chemical components in cosmetics, and relates to a method for detecting the content of retinol and 6 retinol derivatives in cosmetics.
Background
Skin is an important organ for maintaining human life and health, however, it gradually begins to age with the aging and the influence of external factors. With the research and development of cosmetic science and the increasing demand of consumers for the efficacy of cosmetics, anti-aging skin care becomes one of the most important consumer demands.
Retinol (vitamin a) is an essential important substance in life activities, is a kind of micronutrient essential to human body, and plays an important role in maintaining visual health, epithelial cell integrity, participation in growth and reproduction, anti-tumor, maintaining integrity of immune system and the like. Retinol is a fat-soluble vitamin, is unstable to light, heat, acid and alkali, is easily oxidized, and can promote oxidative damage under ultraviolet rays. Retinol can be prepared into retinol ester compounds through chemical reaction, and the common retinol ester compounds comprise retinol acetate, retinol propionate, retinol palmitate, hydroxy pinacolone retinoic acid ester and retinol retinoic acid ester. Retinol and its derivatives act on the skin epidermis and are converted into retinal and further into retinoic acid through endogenous enzymatic reactions on the skin. Retinoic acid is an important growth factor hormone, and can promote cell proliferation and growth, maintain normal skin circulation, repair lost collagen, and reduce signs of wrinkles and premature aging (e.g., abnormal pigmentation, rough spots, and dry skin). Therefore, the retinol and the derivatives thereof have obvious effects on the aspects of anti-aging, acne removal, whitening, oxidation resistance and the like.
The usage of retinol and its derivatives in the cosmetic industry worldwide is on the trend of increasing year by year. The retinol raw materials commonly used in the cosmetic industry mainly include retinol, retinal, retinol acetate, retinol propionate, retinol retinoic acid ester, retinol palmitate, and hydroxy pinacolone retinoic acid ester. Retinoic acid is a relatively irritating drug, and is a prescribed drug that is prohibited for use in cosmetics. Many consumers have recently become enthusiastic to follow the habit of brushing acid or applying vitamin C and vitamin A too often to cause skin intolerance. Retinoids have a strong relative irritation, and intolerant people have the side effects of red swelling, desquamation and the like. In order to meet the pursuit of consumers and the requirements of quick whitening and anti-aging of some products in the market, the retinol and the derivatives thereof are added excessively. How to monitor the quality of the unsmooth cosmetics on the market is increasingly important.
Meanwhile, the retinol and the derivatives thereof have the characteristics of instability, low utilization rate and the like when being applied to cosmetics, and how to stably store the retinol in the cosmetics and slowly release the retinol in the skin is an important problem to be solved urgently by raw material manufacturers. The liposome coating technology is used recently, but the technology has certain problems, such as the preparation process needs to be heated to 40-80 ℃ for heat treatment, the retinol and the derivatives thereof are unstable to heat, and the damage to retinol and other active substances is easily caused in the coating process. How to better control the quality and stability of raw materials of retinol-based wraps is especially important for raw material manufacturers and cosmetic companies.
At present, liquid chromatography detection methods for retinol and derivatives thereof are numerous, for example, HPLC (high performance liquid chromatography) method published by New people in the China and other people for rapidly determining retinol and tocopherol in vegetable oil, food science and technology, 6 th year 2018, mainly aim at detection of retinol in vegetable oil. The HPLC method for measuring the retinol palmitate and the retinol acetate in the infant food with special medical application published by Litao et al, food and machinery, 5 th 2018, mainly aims at detecting the retinol, the retinol palmitate and the retinol acetate in the food. Jingjing et al published in "determination of retinol content in children's serum by reversed-phase high performance liquid chromatography internal standard method", public health and preventive medicine, 2012, 3 rd, mainly performed detection on retinol and retinol palmitate in blood. Due to the large differences between the bases of oils and foods and cosmetics, the above methods may interfere with the testing of cosmetics with respect to bases.
For another example, the content of hydroxy pinacoline retinoic acid ester in cosmetics is determined by high performance liquid chromatography published by fangylin et al, and the detection is mainly performed on hydroxy pinacoline retinoic acid ester in cosmetics in light industrial science and technology, 2021, 2 nd phase. The patent application No. CN201810106756.2 method for measuring vitamin A by high performance liquid chromatography is only the development of a detection method, does not relate to the application of specific products, and the matrix of the cosmetic product may interfere with the method. 9 components such as retinol in cosmetics are determined by high performance liquid chromatography published by Liuyuling et al, daily chemical industry, No. 10 in 2021, the method relates to retinol, retinol acetate, retinol propionate and retinol palmitate, and other 5 forbidden components in cosmetics, and the method has the advantages of long analysis time of 1 hour, unstable baseline and low detection efficiency.
And at present, no detection method is reported for retinol retinoic acid ester. No detection method is reported for simultaneously detecting 7 retinoids.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention provides a method for detecting the content of retinol and 6 retinol derivatives in cosmetics, which is used to solve the problem of the lack of a method for measuring the content of retinol and 6 retinol derivatives in cosmetics and raw materials in the prior art.
In order to achieve the above objects and other related objects, the present invention provides a method for detecting the content of retinol and 6 retinol derivatives in cosmetics, comprising: adding a cosmetic sample into a first solvent for dispersion, adding a second solvent for ultrasonic extraction, filtering, collecting a sample solution obtained from the filtrate, determining the detection wavelength by ultraviolet absorption spectrometry (UV), and performing qualitative and quantitative detection by High Performance Liquid Chromatography (HPLC) to determine the content of retinol and 6 retinol derivatives in the sample solution.
Preferably, the cosmetic sample is selected from one of a serum, a lotion or a cream.
The essence, the emulsion and the cream are all commercially available essence, emulsion and cream, and can be purchased from the market.
The Essence, also called Essence (Essence) or Essence milk, is one of the skin care products for the face, and is usually used after skin water is applied and before the lotion or cream. It contains plant extract, ceramide, squalane, etc., and has antiaging, wrinkle removing, moisture keeping, skin whitening, and speckle removing effects. Specifically, for example, the beauty of blue, Qinqin lotus crystal-clear repair essence produced by the company of Mirabi cosmetics (Wuhan) Ltd; also as the skin barrier repair essence cream of Yuze sun produced by Shanghai's family and United nationality GmbH; and essence of lanocoma muscle bottom repairing and relieving essence produced by Eurya (China) Limited company.
The emulsions (deposition), also known as milks (milk), are used for skin care and are cosmetics intended to maintain a moderate level of moisture in the skin, particularly in the outermost stratum corneum of the skin. It features that it can not only keep the balance of skin water, but also supplement important oily component, hydrophilic moisture-keeping component and water, and can be used as the carrier of active component to be absorbed by skin for regulating and nourishing skin. The skin care lotion concretely comprises skin moistening lotion, skin cleaning lotion, sun protection lotion, hand protection lotion, body lotion and the like. Specifically, for example, the beauty of blue, Qinqin Lianying tough emulsion produced by the company Mirabi beauty products (Wuhan) Ltd; also as the skin barrier repair body lotion of Yuze produced by Shanghai's home-based Union Ltd; further, the emulsion is a Perlya hydrodynamic emulsion produced by Perlya cosmetics Co.
The cream (vanishingcream), namely the skin-care cream, is an emulsified product with the skin-care effect, and can be divided into cream (soft in texture) and cream (hard in texture) according to the appearance state; the oil content can be divided into vanishing cream, neutral cream (skin caring cream) and balm; oil-in-water (O/W) type and composite emulsion type (W/O/W or O/W/O) can be classified according to the emulsion type. Specifically, for example, the beauty of blue, Qinqin Lianying, tough face cream produced by the company Mirabi beauty products (Wuhan) Co; also like koyan high moisturizing cream produced by orliya (china) limited; further, for example, the new multi-effect Zhiyan eye cream of Arselagi produced by Yintai cosmetics (Suzhou) Co.
Preferably, the retinol and the 6 retinol derivatives include 7 types, which are retinol (CAS number 68-26-8), retinal (CAS number 116-31-4), retinol acetate (CAS number 127-47-9), retinol propionate (CAS number 7069-42-3), retinol retinoic acid ester (CAS number 15498-86-9), retinol palmitate (CAS number 79-81-2), and hydroxy pinacolone retinoic acid ester (CAS number 893412-73-2).
Preferably, the ratio of the added mass of the cosmetic sample to the added volume of the first solvent is 1:1-10 g/mL.
Preferably, the first solvent is tetrahydrofuran.
Preferably, the dispersion is dispersed by vortex oscillation, and the oscillation time of the vortex oscillation is 0.5-2.0 min.
Preferably, the second solvent is methanol.
Preferably, the ratio of the added mass of the cosmetic sample to the added volume of the second solvent is 1:15-24 g/mL.
Preferably, the ultrasonic extraction time is 10-20 min.
Preferably, the filtration is performed by using a filter membrane. Preferably, the filter is a 0.20-0.45 μm filter, preferably 0.22 μm.
Preferably, the method for determining the detection wavelength by using an ultraviolet absorption broad spectrum method and then performing qualitative and quantitative detection by using a High Performance Liquid Chromatography (HPLC) comprises the following steps:
1) adding a third solvent into a reference substance of the retinol and the 6 retinol derivatives for dissolving, and then fixing the volume to prepare a mixed reference substance solution of the retinol and the 6 retinol derivatives and 7 single reference substance solutions respectively;
2) respectively detecting the 7 single reference substance solutions in the step 1) by adopting an ultraviolet absorption spectrometry (UV) method, and determining the maximum absorption wavelengths of the retinol and the 6 retinol derivatives;
3) detecting the test solution and the mixed reference solution in the step 1) respectively by adopting a High Performance Liquid Chromatography (HPLC), comparing the retention time for qualitative determination by taking the maximum absorption wavelength determined in the step 2) as a detection wavelength, and determining the contents of the retinol and the 6 retinol derivatives in the test solution by adopting an external standard method for quantitative determination.
Preferably, in step 1), the third solvent is a methanol solution containing tetrahydrofuran.
More preferably, in the methanol solution containing tetrahydrofuran, the volume ratio of the added tetrahydrofuran to the added methanol is 1: 1-50.
Preferably, in step 1), the mixed reference solution and/or the single reference solution is prepared by stepwise dilution.
Preferably, in the step 1), the content of the retinol in the mixed control solution and/or the single control solution is in the range of 5-200 μ g/ml; the content of retinene is 10-400 μ g/ml; the content range of the retinol acetate is 10-400 mug/ml; the content range of the retinol propionate is 5-200 mug/ml; the content range of the retinol retinoic acid ester is 10-400 mug/ml; the content range of the retinol palmitate is 10-400 mug/ml; the content range of the hydroxy pinacolone retinoic acid ester is 10-400 mug/ml.
More preferably, the content of the retinol in the mixed control solution and/or the single control solution is in the range of 5-80 μ g/ml; the content of retinene is 10-160 μ g/ml; the content range of the retinol acetate is 10-160 mug/ml; the content range of the retinol propionate is 5-80 mug/ml; the content range of the retinol retinoic acid ester is 10-160 mug/ml; the content range of the retinol palmitate is 10-160 mug/ml; the content range of the hydroxy pinacolone retinoic acid ester is 10-160 mug/ml.
Preferably, in the step 2), the full scanning wavelength range of the ultraviolet absorption spectroscopy (UV) is 200-800 nm.
Preferably, in the step 2), in the ultraviolet absorption spectrometry (UV), the maximum absorption wavelength λ max of the retinol is 325 nm; the maximum absorption wavelength lambda max of the retinene is 380 nm; the maximum absorption wavelength lambda max of the retinol acetate is 325 nm; the maximum absorption wavelength lambda max of the retinol propionate is 325 nm; the maximum absorption wavelength lambda max of the retinol retinoic acid ester is 340 nm; the maximum absorption wavelength lambda max of the retinol palmitate is 325 nm; the maximum absorption wavelength λ max of said hydroxyppinacolone retinoic acid ester is 360 nm.
Preferably, in the step 3), the detector used in the High Performance Liquid Chromatography (HPLC) is a photodiode array detector (DAD).
Preferably, in the step 3), the chromatographic column in the high performance liquid chromatography is a C18 chromatographic column.
More preferably, the column in the high performance liquid chromatography is an Agilent Eclipse Plus C18 column (column length 250mm, inner diameter 4.6mm, particle size 5 μm).
Preferably, in the step 3), the detection wavelength in the high performance liquid chromatography is 325-380 nm. More preferably, in the high performance liquid chromatography, the detection wavelength of retinol, retinol acetate, retinol propionate, retinol palmitate is 325nm, the detection wavelength of retinol retinoic acid ester is 340nm, the detection wavelength of hydroxy pinacolone retinoic acid ester is 360nm, and the detection wavelength of retinal is 380 nm.
Preferably, in step 3), the column temperature in the high performance liquid chromatography is 25-35 ℃. More preferably, the column temperature in the high performance liquid chromatography is 30 ℃.
Preferably, in the step 3), the flow rate in the high performance liquid chromatography is 0.8-1.2 ml/min. More preferably, the flow rate in the high performance liquid chromatography is 1.0 ml/min.
Preferably, in the step 3), the sample injection amount in the high performance liquid chromatography is 5-15 μ l. More preferably, the sample amount in the high performance liquid chromatography is 10. mu.l.
Preferably, in the step 3), in the high performance liquid chromatography, the mobile phase is selected from one of acetonitrile-tetrahydrofuran or methanol-acetonitrile-tetrahydrofuran, wherein when the mobile phase is acetonitrile-tetrahydrofuran, the phase a is acetonitrile, the phase B is tetrahydrofuran, and the analysis time is 30 min; when the mobile phase is methanol-acetonitrile-tetrahydrofuran, the phase A is methanol, the phase B is acetonitrile, the phase C is tetrahydrofuran, and the analysis time is 35 min; gradient elution.
More preferably, when the mobile phase is acetonitrile-tetrahydrofuran, the specific procedure of the gradient elution is:
0-10 min, phase A: the volume ratio of the phase B is 100: 0-100: 0;
10-12 min, phase A: the volume ratio of the phase B is 100: 0-70: 30, of a nitrogen-containing gas;
12-23 min, phase A: the volume ratio of the phase B is 70: 30-70: 30, of a nitrogen-containing gas;
23-25 min, phase A: the volume ratio of the phase B is 70: 30-100: 0;
25-30 min, phase A: the volume ratio of the phase B is 100: 0-100: 0.
more preferably, when the mobile phase is methanol-acetonitrile-tetrahydrofuran, the specific procedure of the gradient elution is:
0-14min, phase A: phase B: the volume ratio of the phase C is 10:90:0-10:90: 0;
14-16min, phase A: phase B: the volume ratio of the phase C is 10:90:0-10:60: 30;
16-27min, phase A: phase B: the volume ratio of the phase C is 10:60:30-10:60: 30;
27-29min, phase A: phase B: the volume ratio of the phase C is 10:60:30-10:90: 0;
29-35min, phase A: phase B: the volume ratio of the phase C is 10:90:0-10:90: 0.
Preferably, in step 3), the external standard method comprises the following steps:
A) preparing a series of mixed reference substance solutions with different concentrations according to the step 1), respectively carrying out HPLC detection to obtain a linear relation between the chromatographic peak areas of the retinol and the 6 retinol derivatives and the contents of the corresponding retinol and the 6 retinol derivatives, drawing a corresponding standard working curve, and calculating to obtain a regression equation of the standard working curve of the retinol and the 6 retinol derivatives;
B) and (3) carrying out HPLC detection on the test solution, substituting the chromatographic peak areas of the obtained retinol and 6 retinol derivatives into the regression equation of the standard working curve of the corresponding retinol and 6 retinol derivatives in the step A), and calculating to obtain the contents of the retinol and 6 retinol derivatives in the test solution.
More preferably, in the standard working curve, the areas of the chromatographic peaks of retinol and 6 retinol derivatives are taken as the ordinate (Y-axis), and the contents of the corresponding retinol and 6 retinol derivatives are taken as the abscissa (X-axis).
As described above, the method for detecting the contents of retinol and 6 retinol derivatives in cosmetics provided by the present invention can simultaneously perform accurate quantitative qualitative detection on retinol and 6 retinol derivatives (retinol, retinal, retinol acetate, retinol propionate, retinol palmitate, hydroxy pinacolone retinoic acid ester and retinol retinoic acid ester) in cosmetics by using pretreatment under optimized conditions and an instrumental detection method.
The method has the characteristics of simple and convenient pretreatment, reliable sample separation, accurate quantitative determination and the like, and has the advantages of good detection recovery rate, high accuracy, high sensitivity and low detection limit. The method not only meets the simultaneous determination of multiple components of batch samples, but also improves the detection efficiency and reduces the experiment cost, and the establishment of the test method has important significance for the supervision of the market of the retinol cosmetics and the retinol cosmetic raw materials in China.
Under the background of new regulations and regulations of cosmetics, the requirement of a cosmetic formula on the efficacy becomes an important consideration, the formula principle is gradually changed from the previous physicochemical thought to the biochemical thought, the safety problems of irritation and the like of the product can be caused by excessive addition of active substances, and the balance of the active ingredient metering and the product efficacy is very important. The method is helpful for cosmetic enterprises to search raw materials with higher quality, control the balance between the optimal efficacy and the measurement, verify whether the production process is reliable, and ensure the stable efficacy of the raw materials and the products. And meanwhile, a reliable quality inspection means is provided for enterprises to research and develop and produce products.
Drawings
Fig. 1 shows uv spectrograms of retinol and 6 retinol derivatives in the present invention, in which 1: hydroxy pinacolone retinoic acid ester; 2: retinol propionate; 3: retinol acetate; 4: retinol; 5: retinol palmitate; 6: retinol retinoic acid ester; 7: retinal.
Fig. 2 shows standard operating graphs 2a, 2b, 2c, 2d, 2e, 2f, 2g of retinol and 6 retinol derivatives in the present invention, wherein fig. 2a is retinol, fig. 2b is retinal, fig. 2c is retinol acetate, fig. 2d is retinol propionate, fig. 2e is retinol retinoic acid ester, fig. 2f is retinol palmitate, and fig. 2g is hydroxy pinacolone retinoic acid ester.
Fig. 3 shows a high performance liquid chromatogram of retinol and 6 retinol derivatives in the present invention, in which 1: retinol; 2: retinal; 3: retinol acetate; 4: hydroxy pinacolone retinoic acid ester; 5: retinol propionate; 6: retinol retinoic acid ester; 7: retinol palmitate.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The reagents and equipment used in the following examples are as follows:
1. reagent
Acetonitrile, tetrahydrofuran, methanol (purity: chromatographically pure, merck-stockhol, inc.); deionized water (self-made by a water purifier).
Retinol (purity: not less than 98%, Beijing Soilebao Tech & ltd & gt), retinal (purity: not less than 98%, Shanghai-sourced leaf Biotech limited), retinol acetate (purity: 94.5%, Shanghai-sourced leaf Biotech limited), retinol propionate (purity: not less than 98%, Aladdin), retinol palmitate (purity: not less than 96%, Shanghai-sourced leaf Biotech limited sourced leaf organism), hydroxy pinacolone retinoic acid ester (purity: not less than 99%, Shanghai-sourced leaf Biotech limited), retinol retinoic acid ester (purity: not less than 98%, Shanghai-sourced leaf Biotech limited).
2. Instrument for measuring the position of a moving object
Agilent 1260Infinity II high performance liquid chromatograph (Agilent, USA); 1900i UV-Vis spectrometer (Shimadzu, Japan).
Example 1
1. Standard stock solutions
Taking a proper amount of retinol and standard products of 6 retinol derivatives (retinol, retinal, retinol acetate, retinol propionate, retinol palmitate, hydroxy pinacolone retinoic acid ester and retinol retinoic acid ester), accurately measuring the standard products to 0.0001g, placing the standard products in a same 25mL volumetric flask, dissolving the standard products by using a methanol solution containing tetrahydrofuran, measuring the volume to the scale, shaking up the volumetric flask uniformly, and preparing a mixed standard stock solution.
Meanwhile, taking a proper amount of standard substances of retinol and 6 retinol derivatives, accurately measuring the standard substances to 0.0001g, respectively placing the standard substances into 7 25mL volumetric flasks, dissolving the standard substances by using a methanol solution containing tetrahydrofuran, metering the volume to a scale, shaking up, and preparing 7 single standard stock solutions.
In the above methanol solution containing tetrahydrofuran, the volume ratio of tetrahydrofuran to methanol was 2: 23. The mixed standard stock solutions and the 7 single standard stock solutions were stored in a refrigerator at 4 ℃ for refrigeration. The concentrations of each component in the mixed standard stock solution and the 7 single standard stock solutions are shown in table 1.
TABLE 1
2. Control solution
And taking the mixed standard stock solution, adding a methanol solution containing tetrahydrofuran for dilution, and preparing a series of mixed reference substance solutions with different concentrations. Meanwhile, 7 single standard stock solutions are respectively added into methanol solution containing tetrahydrofuran for dilution to respectively prepare 7 single reference substance solutions. In the above methanol solution containing tetrahydrofuran, the volume ratio of tetrahydrofuran to methanol was 2: 23. The concentrations of each component in the combined control solution and the 7 single control solutions are shown in table 2. The reference solution is prepared as it is.
TABLE 2
Example 2
1. Preparation of test solution
Adding 1g (accurate to 0.1mg) of cosmetic sample into 25mL colorimetric tube with plug, adding tetrahydrofuran 5mL, performing vortex oscillation for 1min for dispersion, adding methanol to constant volume to scale, performing ultrasonic extraction for 15min, and filtering part of the liquid with 0.22 μm filter membrane to obtain sample solution No. 1.
2. UV measurement
The 7 single control solutions of example 1 were taken and measured by total scanning with UV absorption spectroscopy in the wavelength range of 200-800 nm. Detecting to obtain the maximum absorption wavelength lambda max of the retinol as 325 nm; the maximum absorption wavelength λ max of retinene is 380 nm; the maximum absorption wavelength λ max of retinol acetate is 325 nm; the maximum absorption wavelength λ max of retinol propionate is 325 nm; the maximum absorption wavelength lambda max of the retinol retinoic acid ester is 340 nm; the maximum absorption wavelength λ max of retinol palmitate is 325 nm; the maximum absorption wavelength λ max of hydroxyppinacolone retinoic acid ester is 360 nm.
3. HPLC determination
The sample solution 1# and the mixed reference solutions with different concentrations in example 1 were respectively detected by High Performance Liquid Chromatography (HPLC), the maximum absorption wavelength of UV measurement was used as the detection wavelength, the retention time was compared and qualitative determination was performed, and quantitative determination was performed by external standard method. The method comprises the steps of respectively carrying out HPLC detection on a series of mixed reference substance solutions with different concentrations to obtain a linear relation between the chromatographic peak areas of the retinol and the 6 retinol derivatives and the contents of the corresponding retinol and the 6 retinol derivatives, drawing a corresponding standard working curve, and calculating to obtain a regression equation of the standard working curve of the retinol and the 6 retinol derivatives. And performing HPLC detection on the sample solution 1#, substituting the chromatographic peak areas of the obtained retinol and 6 retinol derivatives into the regression equation of the standard working curve of the corresponding retinol and 6 retinol derivatives, and calculating to obtain the contents of the retinol and 6 retinol derivatives in the sample solution.
Wherein, the high performance liquid chromatography comprises the following detection conditions:
the detector is a photodiode array detector (DAD); the chromatographic column is an Agilent Eclipse Plus C18 chromatographic column (the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 μm); the detection wavelength of the retinol, the retinol acetate, the retinol propionate and the retinol palmitate is 325nm, the detection wavelength of the retinol retinoic acid ester is 340nm, the detection wavelength of the hydroxy pinacolone retinoic acid ester is 360nm, and the detection wavelength of the retinal is 380 nm; the column temperature is 30 ℃; the flow rate is 1.0 ml/min; the sample amount is 10 mul; the mobile phase is acetonitrile-tetrahydrofuran, wherein the phase A is acetonitrile, and the phase B is tetrahydrofuran; the analysis time is 30 min; gradient elution.
The specific procedure for gradient elution was:
0-10 min, phase A: the volume ratio of the phase B is 100: 0-100: 0;
10-12 min, phase A: the volume ratio of the phase B is 100: 0-70: 30, of a nitrogen-containing gas;
12-23 min, phase A: the volume ratio of the phase B is 70: 30-70: 30, of a nitrogen-containing gas;
23-25 min, phase A: the volume ratio of the phase B is 70: 30-100: 0;
25-30 min, phase A: the volume ratio of the phase B is 100: 0-100: 0.
example 3
1. Preparation of test solution
Adding 1g (accurate to 0.1mg) of cosmetic sample into 25mL colorimetric tube with plug, adding tetrahydrofuran 4mL, performing vortex oscillation for 1.5min for dispersion, adding methanol to constant volume to scale, performing ultrasonic extraction for 12min, and filtering part of liquid with 0.22 μm filter membrane to obtain sample solution 2 #.
2. UV measurement
The maximum absorption wavelengths of retinol and 6 retinol derivatives were determined as in step 2 of example 2.
3. HPLC determination
The sample solution 2# and a series of mixed reference solutions with different concentrations in example 1 were respectively detected by High Performance Liquid Chromatography (HPLC), the maximum absorption wavelength of UV measurement was used as the detection wavelength, the retention time was compared and qualitative determination was performed, and quantitative determination was performed by external standard method. The method comprises the steps of respectively carrying out HPLC detection on a series of mixed reference substance solutions with different concentrations to obtain a linear relation between the chromatographic peak areas of the retinol and the 6 retinol derivatives and the contents of the corresponding retinol and the 6 retinol derivatives, drawing a corresponding standard working curve, and calculating to obtain a regression equation of the standard working curve of the retinol and the 6 retinol derivatives. And performing HPLC detection on the sample solution 2#, substituting the chromatographic peak areas of the obtained retinol and 6 retinol derivatives into the regression equation of the standard working curve of the corresponding retinol and 6 retinol derivatives, and calculating to obtain the contents of the retinol and 6 retinol derivatives in the sample solution.
Wherein, the high performance liquid chromatography comprises the following detection conditions:
the detector is a photodiode array detector (DAD); the chromatographic column is an Agilent Eclipse Plus C18 chromatographic column (the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 μm); the detection wavelength of the retinol, the retinol acetate, the retinol propionate and the retinol palmitate is 325nm, the detection wavelength of the retinol retinoic acid ester is 340nm, the detection wavelength of the hydroxy pinacolone retinoic acid ester is 360nm, and the detection wavelength of the retinal is 380 nm; the column temperature is 30 ℃; the flow rate is 1.0 ml/min; the sample amount is 10 mul; the mobile phase is methanol-acetonitrile-tetrahydrofuran, wherein the phase A is methanol, the phase B is acetonitrile, and the phase C is tetrahydrofuran; the analysis time is 35 min; gradient elution.
The specific procedure for gradient elution was:
0-14min, phase A: phase B: the volume ratio of the phase C is 10:90:0-10:90: 0;
14-16min, phase A: phase B: the volume ratio of the phase C is 10:90:0-10:60: 30;
16-27min, phase A: phase B: the volume ratio of the phase C is 10:60:30-10:60: 30;
27-29min, phase A: phase B: the volume ratio of the phase C is 10:60:30-10:90: 0;
29-35min, phase A: phase B: the volume ratio of the phase C is 10:90:0-10:90: 0.
Example 4
A series of mixed control solutions of different concentrations were prepared as in example 1, and the concentrations of the components in the control solutions are shown in Table 2. According to the HPLC conditions of step 3 in example 2, 10 μ l of the mixed control solution was precisely aspirated and injected into a liquid chromatograph, the mixed control concentration (μ g/ml) was plotted with the abscissa and the ordinate as the peak area, and the standard regression equation, correlation coefficient and linear range of retinol and 6 retinol derivatives were determined and calculated, and the specific results are shown in Table 3. As is clear from Table 3, retinol and 6 retinol derivatives showed good linear relationship in the concentration range examined.
TABLE 3
| Compound (I) | Linear equation of equations | Coefficient of correlation R2 | Linear Range (. mu.g.mL)-1) |
| Retinol | y＝29.561x-14.029 | 0.9997 | 5-80 |
| Retinoic aldehyde | y＝75.307x-38.208 | 1 | 10-160 |
| Retinol acetic acid ester | y＝18.118x-10.934 | 0.9998 | 10-160 |
| Retinol propionate | y＝71.561x-26.664 | 0.9998 | 5-80 |
| Retinol | y＝25.807x–4.4583 | 0.9997 | 10-160 |
| Retinol palmitate | y＝37.167x–72.483 | 0.9999 | 10-160 |
| Hydroxy pinacolone retinoic acid ester | y＝69.71x–32.001 | 0.9999 | 10-160 |
At the same time, the mixed control solution was diluted and measured under the HPLC conditions of step 3 in example 2, and a signal three times as a noise was used as a detection limit to obtain retinol 1.0. mu.g.mL-1Retinene 1.0. mu.g/mL-1Retinol acetate 1.0. mu.g/mL-10.5. mu.g/mL of retinol propionate-1Retinol retinoic acid ester 2.0. mu.g/mL-12.0. mu.g/mL of retinol palmitate-11.0. mu.g/mL of hydroxyppinacolone retinoic acid ester-1(ii) a Taking ten times of noise signals as a quantitative limit to obtain 3.0 mu g/mL of retinol-13.0. mu.g/mL of retinal-1Retinol acetate 3.0. mu.g/mL-11.5. mu.g/mL of retinol propionate-1Retinol retinoic acid ester 6.0. mu.g/mL-1Retinol palmitic acidEster 6.0. mu.g/mL-13.0. mu.g/mL of hydroxyppinacolone retinoic acid ester-1. The specific data are shown in Table 4.
TABLE 4
| Component name | Detection Limit (μ g. mL)-1) | Limit of quantitation (μ g. mL)-1) |
| Retinol | 1.0 | 3.0 |
| Retinoic aldehyde | 1.0 | 3.0 |
| Retinol acetic acid ester | 1.0 | 3.0 |
| Retinol propionate | 0.5 | 1.5 |
| Retinol | 2.0 | 6.0 |
| Retinol palmitate | 2.0 | 6.0 |
| Hydroxy pinacolone retinoic acid ester | 1.0 | 3.0 |
Example 5
7 single control solutions were taken as in example 1, and retinol in the single control solution was 20. mu.g.mL-1Retinaldehyde 40. mu.g/mL-1Retinol acetate 40. mu.g/mL -120. mu.g/mL of retinol propionate-1Retinol retinoic acid ester 40. mu.g/mL-1Retinol palmitate 40. mu.g/mL-140. mu.g/mL of hydroxyppinacolone retinoic acid ester -17 test solutions were prepared as in step 1 of example 2. Precisely absorbing 10 μ l of the same sample solution according to the HPLC conditions of step 3 in example 2, injecting into a liquid chromatograph, repeating the sample injection analysis for 6 times, measuring the peak area of each component, and finding the specific precision results shown in Table 5. The results show that the RSDs of retinol and 6 retinol derivatives are retinol: 1.76%, retinal: 0.42%, retinol acetate: 0.77%, retinol propionate: 0.73%, retinol palmitate: 1.06%, retinol retinoic acid ester: 1.56%, hydroxy pinacolone retinoic acid ester: 0.42 percent. The chromatographic peak areas RSD are all less than 2%, which indicates that the precision of the instrument is good.
TABLE 5
Example 6
Selecting essence containing no 7 retinol and its derivatives [ Ulian blue beauty essence from Ulian beauty products (Wuhan) Co., Ltd.)]Skin care lotion [ Mitsui Yisi cosmetics (Wuhan) Co., Ltd. ] Ulsan blue beauty and Qinqian Lianying tough lotion]'Mian' beauty of blue-blue Lian Yinqi face cream produced by Mian Yisi cosmetics (Wuhan) Co Ltd]3 kinds of cosmetics with different matrixes,2 portions of each, 1g of each (to the nearest 0.0001g), are respectively added into a 10mL colorimetric tube with a plug, the mixed standard stock solution in the example 1 is added, and a methanol solution containing tetrahydrofuran with the volume ratio of 2:23 is added to the volume to be calibrated to prepare 10 mu g/mL of retinol-1Retinaldehyde 40. mu.g/mL-1Retinol acetate 40. mu.g/mL-1Retinol propionate 40. mu.g/mL-1Retinol retinoic acid ester 40. mu.g/mL -120. mu.g/mL of retinol palmitate-140. mu.g/mL of hydroxyppinacolone retinoic acid ester-1The test solution was prepared in step 1 of example 2. 6 parts of the same test sample solution are taken, 10 mul of the test sample solution is precisely absorbed and injected into a liquid chromatograph, sample injection analysis is respectively carried out according to the HPLC conditions of the step 3 in the embodiment 2, the peak area is measured, and specific repeatability results are shown in a table 6.
The results show that the RSDs of retinol and 6 retinol derivatives are retinol: 2.24%, retinal: 1.42 percent; retinol acetate: 0.97%, retinol propionate: 0.40%, retinol retinoic acid ester: 1.06%, retinol palmitate: 2.21%, hydroxy pinacolone retinoic acid ester: 0.98 percent. The chromatographic peak areas RSD are all less than 3%, which shows that the method has good repeatability.
TABLE 6
Example 7
Selecting essence containing no 7 retinol and derivatives thereof [ Meiqin lotus Fluoresceration essence of blue produced by Mitsui beauty products (Wuhan) Co., Ltd ], emulsion [ Meiqin lotus Fluoresceration emulsion of blue produced by Mitsui beauty products (Wuhan) Co., Ltd ], cream [ Meiqin lotus Fluoresceration strong cream of blue produced by Mitsui beauty products (Wuhan) Co., Ltd ], 3 cosmetics with different matrixes, taking 9 parts of each cream, taking 1g (accurate to 0.0001g) of each cream, adding 0.25mL/0.5mL/1.0mL of the mixed standard stock solution in example 1 into a 10mL color comparison tube, adding 3 samples of three groups of concentrations in parallel, adding 3mL of tetrahydrofuran, dispersing by swirling for 1min, adding methanol to fix the volume to scale, performing ultrasonic extraction for 15min, and recovering the theoretical addition amount of 3 samples of standard samples, retinol, retinol propionic acid
The esters were 5. mu.g.mL, respectively-1、10μg·mL-1、20μg·mL-1Retinaldehyde, retinol acetate, retinol retinoic acid ester, retinol palmitate, and hydroxy pinacolone retinoic acid ester were 10. mu.g.mL, respectively-1、20μg·mL-1、40μg·mL-1And filtering part of the liquid through a 0.22 mu m filter membrane.
Then, 10. mu.l of the sample was precisely pipetted and injected into a liquid chromatograph under the HPLC conditions of step 3 in example 2, and the results of sample injection analysis were shown in Table 7. As is clear from Table 7, the recovery rates of retinol and 6 retinol derivatives ranged from 92.37-112.0%. The results show that the recovery rates of low, medium and high retinol and 6 derivatives are good, and the precision (RSD) of the method is 0.20-6.14%.
RSD results show that the method has high accuracy. The test result is very close to the actual addition concentration, and the test requirement can be met.
TABLE 7
In conclusion, the method for detecting the content of the retinol and the 6 retinol derivatives in the cosmetics has the characteristics of simple and convenient pretreatment, reliable sample separation, accurate quantitative determination and the like, and has the advantages of good detection recovery rate, high accuracy, high sensitivity and low detection limit. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A method for detecting the content of retinol and 6 retinol derivatives in cosmetics comprises the following steps: adding a cosmetic sample into a first solvent for dispersion, adding a second solvent for ultrasonic extraction, filtering, collecting a sample solution obtained from the filtrate, determining the detection wavelength by ultraviolet absorption spectrometry, and performing qualitative and quantitative detection by high performance liquid chromatography to determine the content of retinol and 6 retinol derivatives in the sample solution.
2. The method for detecting the content of retinol and 6 retinol derivatives in the cosmetic as claimed in claim 1, wherein the cosmetic sample is selected from one of essence, lotion or cream.
3. The method for detecting the content of retinol and 6 retinol derivatives in the cosmetic according to claim 1, which comprises any one or more of the following conditions:
A1) the ratio of the added mass of the cosmetic sample to the added volume of the first solvent is 1:1-10, g/mL;
A2) the first solvent is tetrahydrofuran;
A3) the dispersion adopts vortex oscillation for dispersion, and the oscillation time of the vortex oscillation is 0.5-2.0 min;
A4) the second solvent is methanol;
A5) the ratio of the added mass of the cosmetic sample to the added volume of the second solvent is 1:15-24 g/mL;
A6) the ultrasonic extraction time is 10-20 min;
A7) the filtration adopts a filter membrane for filtration.
4. The method for detecting the content of retinol and 6 retinol derivatives in the cosmetic as claimed in claim 1, wherein the method comprises the following steps:
1) adding a third solvent into a reference substance of the retinol and the 6 retinol derivatives for dissolving, and then fixing the volume to prepare a mixed reference substance solution of the retinol and the 6 retinol derivatives and 7 single reference substance solutions respectively;
2) respectively detecting the 7 single reference substance solutions in the step 1) by adopting an ultraviolet absorption spectrometry, and determining the maximum absorption wavelength of the retinol and the 6 retinol derivatives;
3) detecting the test solution and the mixed reference solution in the step 1) by adopting a high performance liquid chromatography, comparing the maximum absorption wavelength determined in the step 2) as a detection wavelength, performing qualitative determination, and determining the contents of the retinol and the 6 retinol derivatives in the test solution by adopting an external standard method for quantification.
5. The method for detecting the content of retinol and 6 retinol derivatives in the cosmetic according to claim 4, wherein in the step 1), the third solvent is a methanol solution containing tetrahydrofuran.
6. The method for detecting the content of retinol and 6 retinol derivatives in the cosmetic as claimed in claim 4, wherein the full scanning wavelength range of the UV absorption spectrum method in step 2) is 200-800 nm.
7. The method for detecting the content of retinol and 6 retinol derivatives in the cosmetic as claimed in claim 4, wherein in the step 3), the detection conditions of the high performance liquid chromatography are as follows: the detector is a photodiode array detector; the chromatographic column is a C18 chromatographic column; the detection wavelength is 325-380 nm; the mobile phase is selected from one of acetonitrile-tetrahydrofuran or methanol-acetonitrile-tetrahydrofuran, wherein when the mobile phase is acetonitrile-tetrahydrofuran, the phase A is acetonitrile, the phase B is tetrahydrofuran, and the analysis time is 30 min; when the mobile phase is methanol-acetonitrile-tetrahydrofuran, the phase A is methanol, the phase B is acetonitrile, the phase C is tetrahydrofuran, and the analysis time is 35 min; gradient elution.
8. The method for detecting the content of retinol and 6 retinol derivatives in the cosmetic according to claim 7, wherein when the mobile phase is acetonitrile-tetrahydrofuran, the specific procedure of gradient elution is as follows:
0-10 min, phase A: the volume ratio of the phase B is 100: 0-100: 0;
10-12 min, phase A: the volume ratio of the phase B is 100: 0-70: 30, of a nitrogen-containing gas;
12-23 min, phase A: the volume ratio of the phase B is 70: 30-70: 30, of a nitrogen-containing gas;
23-25 min, phase A: the volume ratio of the phase B is 70: 30-100: 0;
25-30 min, phase A: the volume ratio of the phase B is 100: 0-100: 0.
9. the method for detecting the content of retinol and 6 retinol derivatives in the cosmetic according to claim 7, wherein when the mobile phase is methanol-acetonitrile-tetrahydrofuran, the specific procedure of gradient elution is as follows:
0-14min, phase A: phase B: the volume ratio of the phase C is 10:90:0-10:90: 0;
14-16min, phase A: phase B: the volume ratio of the phase C is 10:90:0-10:60: 30;
16-27min, phase A: phase B: the volume ratio of the phase C is 10:60:30-10:60: 30;
27-29min, phase A: phase B: the volume ratio of the phase C is 10:60:30-10:90: 0;
29-35min, phase A: phase B: the volume ratio of the phase C is 10:90:0-10:90: 0.
10. The method for detecting the content of retinol and 6 retinol derivatives in the cosmetic as claimed in claim 4, wherein in step 3), the HPLC further comprises any one or more of the following detection conditions:
B1) the column temperature is 25-35 ℃;
B2) the flow rate is 0.8-1.2 ml/min;
B3) the sample injection amount is 5-15 mul.
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