CN114324219B - 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 contents of retinol and 6 retinol derivatives in cosmetics, which comprises the following steps: adding a cosmetic sample into a first solvent for dispersion, then adding a second solvent for ultrasonic extraction, filtering, taking a sample solution obtained from the filtrate, determining the detection wavelength by adopting an ultraviolet absorption spectrometry, and then carrying out qualitative and quantitative detection by adopting a high performance liquid chromatography to determine the contents of retinol and 6 retinol derivatives in the sample solution. The method for detecting the contents of retinol and 6 retinol derivatives in the cosmetics has the characteristics of simple pretreatment, reliable sample separation, accurate quantitative and qualitative properties, 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 contents of retinol and 6 retinol derivatives in cosmetics.
Background
Skin is an important organ that maintains human life and health, however, as age increases and external factors affect, skin gradually begins to age. With research development of cosmetic science and increasing demands of consumers for cosmetic efficacy, anti-aging skin care becomes one of the most important consumer appeal.
Retinol (vitamin a) is an essential substance in life activities, is a type of micronutrient essential to the human body, and plays an important role in maintaining visual health, integrity of epithelial cells, participation in growth reproduction, anti-tumor, maintenance of the integrity of the 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. The retinol can be prepared into retinol esters by chemical reaction, and common retinol esters include retinol acetate, retinol propionate, retinol palmitate, hydroxy pinacolin retinoate and retinol retinoate. Retinol and its derivatives act on the epidermis of the skin and are converted to retinal, and further retinoic acid, by endogenous enzymatic reactions on the skin. Retinoic acid is an important hormone-like growth factor that promotes cell proliferation and growth, maintains normal circulation of the skin, repairs lost collagen, and reduces signs of wrinkles and premature aging (such as abnormal pigmentation, rough spots, and dry skin). Therefore, the retinol and the derivatives thereof have remarkable effects on anti-aging, acne removal, whitening, antioxidation and the like.
The use of retinol and its derivatives in the global cosmetic industry is on the rise year by year. The materials of retinol commonly used in cosmetic industry mainly comprise retinol, retinaldehyde, retinol acetate, retinol propionate, retinol retinoic acid ester, retinol palmitate, and hydroxy pinacol retinoic acid ester. Retinoic acid has high irritation, and is a prescription drug, and can not be used in cosmetics. Many consumers recently are enthusiastic to follow the skin care habits of acid brushing or early C late a, and improper or excessive use of vitamin C and vitamin a may result in intolerance of the skin. The retinol products have stronger relative irritation, and intolerant people can have side effects such as red swelling, desquamation and the like. Some products on the market have the condition of excessive addition of retinol and its derivatives in order to meet the requirements of consumers for quick whitening and anti-aging. How to monitor the quality of cosmetics which are good and bad in the market is more and more important.
Meanwhile, the retinol and its derivatives have the characteristics of instability, low utilization rate and the like in the application of 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. More recently, liposome encapsulation technology has been popular, but this technology also has a certain problem, such as heating to 40-80 ℃ for heat treatment in the preparation process, retinol and its derivatives are very unstable to heat, and easily cause damage to active substances such as retinol during encapsulation. How to better control the quality and stability of the retinol wrap raw material is particularly important for raw material manufacturers and cosmetic companies.
At present, a plurality of liquid chromatography detection methods are aimed at retinol and derivatives thereof, for example, the HPLC method for rapidly detecting retinol and tocopherol in vegetable oil published by new mushroom et al, food technology, and 2018, stage 6, mainly aims at detecting retinol in vegetable oil. Li Tao et al, HPLC method for determining retinol palmitate and retinol acetate in infant formula for special medical use, food and machinery, 2018, stage 5, mainly for detecting retinol, retinol palmitate and retinol acetate in food. The method disclosed by Jing et al, which is used for measuring the retinol content in children serum by an internal standard method of reverse-phase high performance liquid chromatography, is used for public health and preventive medicine, and mainly aims at detecting retinol and retinol palmitate in blood in the 3 rd stage of 2012. The above methods may interfere with the testing of cosmetics due to the large differences in the bases of oils and foods and cosmetics.
For another example, fang Yilin et al, high performance liquid chromatography, determine the amount of hydroxy pinacolone retinoic acid ester in a cosmetic, light industry technology, 2021, stage 2, mainly detects hydroxy pinacolone retinoic acid ester in cosmetics. Patent application number CN201810106756.2, "method for measuring vitamin a by high performance liquid chromatography", is only a development of a detection method, and does not relate to application of a specific product, and the substrate of the cosmetic product may interfere with the method. Liu Yuling et al, "high Performance liquid chromatography for determination of 9 components such as retinol in cosmetics", daily chemical industry, 2021, phase 10, which involves retinol, retinol acetate, retinol propionate and retinol palmitate, and other 5 components disabled in cosmetics, which has a long analysis time of up to 1 hour, unstable baseline, and low detection efficiency.
Moreover, no detection method has been reported for retinol retinyl esters. The detection method for simultaneously detecting 7 retinol substances is not reported.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a method for detecting the contents of retinol and 6 retinol derivatives in cosmetics, which is used for solving the problem of lack of a method for measuring the contents of retinol and 6 retinol derivatives in cosmetics and raw materials in the prior art.
To achieve the above and other related objects, the present invention provides a method for detecting contents of retinol and 6 retinol derivatives in cosmetics, comprising: adding a cosmetic sample into a first solvent for dispersion, then adding a second solvent for ultrasonic extraction, filtering, taking a sample solution obtained from the filtrate, determining the detection wavelength by adopting an ultraviolet absorption spectrometry (UV), and then carrying out qualitative and quantitative detection by adopting a High Performance Liquid Chromatography (HPLC) to determine the contents of retinol and 6 retinol derivatives in the sample solution.
Preferably, the cosmetic sample is selected from one of essence, emulsion or 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 known as Essence (Essence) or cream, is one of the skin care products for the face, usually used after the lotion or before the cream. The plant extract, ceramide, squalane and other effective components are contained, and the plant extract has the effects of resisting aging, resisting wrinkle, preserving moisture, whitening, removing spots and the like. Specifically, for example, the Mei Qin Lian Ying moisturizing and repairing essence of the fruit blue produced by the company of Uygur autonomous cosmetic (Wuhan); the jade skin barrier repair essence milk also is manufactured by Shanghai household United stock Co., ltd; such as the essence of the lankoog skin-bottom repairing and relieving essence produced by Europea (China) limited company.
The emulsion (condition), also called milk (milk), is a cosmetic product used for skin care with the aim of maintaining moderate moisture in the skin, in particular in the outermost stratum corneum of the skin. It features that it can not only maintain the balance of skin moisture, but also supplement important oily components, hydrophilic moisture-keeping components and moisture, and can be used as carrier of active component for skin absorption, so as to attain the goal of conditioning and nourishing skin. The skin care lotion comprises skin care lotion, skin cleaning lotion, sun protection lotion, hand protection lotion, body lotion and the like. Specifically, for example, the emulsion is produced by Uo Yisi cosmetics (Wuhan Co., ltd.); the skin barrier of jade zebra also produced by Shanghai household United stock Co., ltd; such as Peratian hydrodynamic moisturizing milk produced by Peratian cosmetics Co., ltd.
The cream (vanishingstream) is an emulsified product with skin care effect, and can be in cream (soft texture) and cream (hard texture) according to appearance state; depending on the oil content, vanishing cream, neutral cream (skin cream) and balsam can be used; oil-in-water (O/W) and composite emulsion (W/O/W or O/W/O) can be distinguished according to the type of the emulsifier. Specifically, for example, the Mei Qin Lian Ying moisturizing and strengthening face cream of the fruit blue produced by the company of Uygur autonomous cosmetic (Wuhan); such as Keyan high moisturizing cream manufactured by Eudragit (China) limited company; such as the new multi-effect intelligent eye cream of the Yashilan Dai produced by the limited company of the Ying Te Li cosmetics (Suzhou).
Preferably, the retinol and 6 retinol derivatives comprise 7 types, namely retinol (CAS No. 68-26-8), retinal (CAS No. 116-31-4), retinol acetate (CAS No. 127-47-9), retinol propionate (CAS No. 7069-42-3), retinol palmitate (CAS No. 15498-86-9), retinol palmitate (CAS No. 79-81-2) and hydroxy pinacol retinol ester (CAS No. 893412-73-2).
Preferably, the ratio of the mass of the cosmetic sample addition to the volume of the first solvent addition is 1:1-10, g/mL.
Preferably, the first solvent is tetrahydrofuran.
Preferably, the dispersion adopts vortex oscillation to disperse, and the oscillation time of the vortex oscillation is 0.5-2.0min.
Preferably, the second solvent is methanol.
Preferably, the ratio of the mass of the cosmetic sample addition to the volume of the second solvent addition is 1:15-24, g/mL.
Preferably, the ultrasonic extraction time is 10-20min.
Preferably, the filtration is performed using a filter membrane. Preferably, the filter is a 0.20-0.45 μm filter, preferably 0.22 μm.
Preferably, the detection wavelength is determined by ultraviolet absorption broad spectrum method, and then qualitative and quantitative detection is performed by High Performance Liquid Chromatography (HPLC), comprising the following steps:
1) Adding a third solvent into the reference substances of the retinol and the 6 retinol derivatives for dissolution, and then fixing the volume to prepare mixed reference substance solutions of the retinol and the 6 retinol derivatives and 7 single reference substance solutions respectively;
2) Detecting 7 single reference substance solutions in the step 1) respectively by adopting an ultraviolet absorption spectrometry (UV), and determining the maximum absorption wavelength of retinol and 6 retinol derivatives;
3) And (2) respectively detecting the sample solution and the mixed reference substance solution in the step (1) 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 the detection wavelength, and determining the contents of retinol and 6 retinol derivatives in the sample 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 tetrahydrofuran to methanol is 1:1-50.
Preferably, in step 1), the mixed reference solution and/or the single reference solution is diluted stepwise.
Preferably, in the step 1), the content of retinol in the mixed reference substance solution and/or the single reference substance solution is 5-200 mug/ml; the content of the retinol is in the range of 10-400 mug/ml; the content of retinol acetate is in the range of 10-400 μg/ml; the content of retinol propionate is in the range of 5-200 μg/ml; the retinol retinoic acid ester content is in the range of 10-400 μg/ml; the content of retinol palmitate is in the range of 10-400 mug/ml; the content of hydroxy pinacolone retinoic acid ester is in the range of 10-400 μg/ml.
More preferably, the content of retinol in the mixed reference solution and/or the single reference solution is in the range of 5-80 mug/ml; the content of the retinol is in the range of 10-160 mug/ml; the content of retinol acetate is in the range of 10-160 μg/ml; the content of retinol propionate is 5-80 μg/ml; the retinol retinoic acid ester content is in the range of 10-160 μg/ml; the content of retinol palmitate is in the range of 10-160 mug/ml; the content of hydroxy pinacolone retinoic acid ester is 10-160 μg/ml.
Preferably, in step 2), the full scan wavelength range of the ultraviolet absorption spectroscopy (UV) is 200-800nm.
Preferably, in step 2), the maximum absorption wavelength λmax=325 nm of the retinol in the ultraviolet absorption spectrometry (UV); the maximum absorption wavelength λmax=380 nm of said retinoid; the maximum absorption wavelength of retinol acetate λmax=325 nm; the maximum absorption wavelength λmax=325 nm of the retinol propionate; the maximum absorption wavelength λmax=340 nm of the retinol retinoic acid ester; the maximum absorption wavelength λmax=325 nm of said retinol palmitate; the maximum absorption wavelength λmax=360 nm of the hydroxy pinacolone retinoic acid ester.
Preferably, in step 3), the detector used in the High Performance Liquid Chromatography (HPLC) is a photodiode array detector (DAD).
Preferably, in step 3), the chromatographic column in the high performance liquid chromatography is a C18 chromatographic column.
More preferably, the chromatographic column in the high performance liquid chromatography is a Agilent Eclipse Plus C18 chromatographic column (column length of 250mm, inner diameter of 4.6mm, particle size of 5 μm).
Preferably, in step 3), the detection wavelength in the high performance liquid chromatography is 325-380nm. 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 pinacol retinoic acid ester is 360nm, and the detection wavelength of retinal is 380nm.
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 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 1.0ml/min.
Preferably, in step 3), the sample injection amount in the high performance liquid chromatography is 5-15 μl. More preferably, the sample injection amount in the high performance liquid chromatography is 10 μl.
Preferably, in 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 30min; when the mobile phase is methanol-acetonitrile-tetrahydrofuran, the A phase is methanol, the B phase is acetonitrile, the C phase is tetrahydrofuran, and the analysis time is 35min; gradient elution.
More preferably, when the mobile phase is acetonitrile-tetrahydrofuran, the specific procedure for 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;
12-23 min, phase A: the volume ratio of the phase B is 70:30-70:30;
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 for the gradient elution is:
0-14min, phase A: and B phase: the volume ratio of the phase C is 10:90:0-10:90:0;
14-16min, phase A: and B phase: the volume ratio of the phase C is 10:90:0-10:60:30;
16-27min, phase A: and B phase: the volume ratio of the phase C is 10:60:30-10:60:30;
27-29min, phase A: and B phase: the volume ratio of the phase C is 10:60:30-10:90:0;
29-35min, phase A: and B phase: 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 performing HPLC detection to obtain linear relations between chromatographic peak areas of retinol and 6 retinol derivatives and contents of the corresponding retinol and 6 retinol derivatives, drawing corresponding standard working curves, and calculating to obtain regression equations of the standard working curves of the retinol and 6 retinol derivatives;
b) And (3) carrying out HPLC detection on the sample solution, substituting the chromatographic peak areas of the obtained retinol and 6 retinol derivatives into a regression equation of the standard working curves 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 sample solution.
More preferably, the standard working curve is plotted on the ordinate (Y-axis) against the areas of the peaks of the retinol and 6 retinol derivatives, and the content of the corresponding retinol and 6 retinol derivatives is plotted on the abscissa (X-axis).
As described above, the method for detecting the contents of retinol and 6 retinol derivatives in cosmetics provided by the invention can be used for simultaneously carrying out accurate quantitative qualitative detection on the retinol and 6 retinol derivatives (retinol, retinaldehyde, retinol acetate, retinol propionate, retinol palmitate, hydroxy pinacol retinoic acid ester and retinol retinoic acid ester) in cosmetics by adopting pretreatment with optimized conditions and an instrument detection method.
The method has the characteristics of simple pretreatment, reliable sample separation, accurate quantitative and qualitative properties, 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 measurement of multiple components of batch samples, but also improves the detection efficiency and reduces the experimental cost, and the establishment of the test method has important significance for the supervision of domestic retinol cosmetics and the market of retinol cosmetic raw materials.
Under the background of new laws of cosmetics, the requirement of the cosmetic formula on efficacy becomes an important consideration, the formula principle is gradually changed from the previous physicochemical thinking to the biochemical thinking, the excessive addition of active substances can cause the safety problems such as irritation and the like of the product, and the balance of the metering of active ingredients and the efficacy of the product is particularly important. The method is helpful for cosmetic enterprises to find raw materials with higher quality, balance between the optimal control effect and metering, verify whether the production process is reliable, and ensure the stable effects of the raw materials and the products. Meanwhile, a reliable quality inspection means is provided for enterprises in research and development and production of products.
Drawings
FIG. 1 shows an ultraviolet spectroscopic scan of retinol and 6 retinol derivatives in the present invention, wherein 1: hydroxy pinacolone retinoic acid ester; 2: retinol propionate; 3: retinol acetate; 4: retinol; 5: retinol palmitate; 6: retinol retinoic acid esters; 7: retinaldehyde.
FIG. 2 shows standard working curves 2a, 2b, 2c, 2d, 2e, 2f, 2g for retinol and 6 retinol derivatives in the present invention, wherein FIG. 2a is retinol, FIG. 2b is retinaldehyde, 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 pinacol retinoic acid ester.
FIG. 3 shows a high performance liquid chromatogram of retinol and 6 retinol derivatives in the present invention, wherein 1: retinol; 2: retinaldehyde; 3: retinol acetate; 4: hydroxy pinacolone retinoic acid ester; 5: retinol propionate; 6: retinol retinoic acid esters; 7: retinol palmitate.
Detailed Description
The invention is further illustrated below in connection with specific examples, which are to be understood as being illustrative of the invention and not limiting the scope of the invention.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The reagents and instrumentation used in the following examples were as follows:
1. reagent(s)
Acetonitrile, tetrahydrofuran, methanol (purity: chromatographic purity, merck, inc.); deionized water (self-made by water purifier).
Retinol (purity: 98% or more, beijing Soy Corp.Technical Co., ltd.), retinal (purity: 98% or more, shanghai Source Biotechnology Co., ltd.), retinol acetate (purity: 94.5%, shanghai Meilin Biotechnology Co., ltd.), retinol propionate (purity: 98% or more, altin), retinol palmitate (purity: 96% or more, shanghai Source Biotechnology Co., ltd.), hydroxy pinacol retinol (purity: 99% or more, shanghai Source Biotechnology Co., ltd.), retinol (purity: 98% or more, shanghai Meilin Biotechnology Co., ltd.).
2. Instrument for measuring and controlling the intensity of light
Agilent 1260 Infinicity II high performance liquid chromatograph (Agilent Co., USA); 1900i ultraviolet visible spectrometer (Shimadzu corporation, japan).
Example 1
1. Standard stock solution
Taking appropriate amounts of retinol and 6 retinol derivatives (retinol, retinaldehyde, retinol acetate, retinol propionate, retinol palmitate, hydroxy pinacol retinoate and retinol retinoate) standard substances, accurately measuring 0.0001g, placing into a same 25mL volumetric flask, dissolving with methanol solution containing tetrahydrofuran, fixing volume to scale, shaking, and preparing into mixed standard stock solution.
Meanwhile, taking a proper amount of standard substances of retinol and 6 retinol derivatives, accurately putting the standard substances into 7 25mL volumetric flasks to be respectively accurate to 0.0001g, dissolving the standard substances by using a methanol solution containing tetrahydrofuran, then fixing the volume to scales, and shaking the standard substances uniformly to prepare 7 single standard stock solutions.
In the tetrahydrofuran-containing methanol solution, the ratio of the added volume of tetrahydrofuran to the added volume of methanol is 2:23. The mixed standard stock solution and 7 single standard stock solutions were placed in a refrigerator at 4 ℃ for cold storage. The concentrations of the components in the mixed standard stock solution and the 7 single standard stock solutions are shown in table 1.
TABLE 1
2. Reference substance solution
And (3) taking the mixed standard stock solution, adding a methanol solution containing tetrahydrofuran, diluting, 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, and 7 single reference substance solutions are respectively prepared. In the tetrahydrofuran-containing methanol solution, the ratio of the added volume of tetrahydrofuran to the added volume of methanol is 2:23. The concentrations of the components in the mixed control solution and the 7 single control solutions are shown in Table 2. The reference substance solution is prepared at present.
TABLE 2
Example 2
1. Preparation of test solutions
1g (accurate to 0.1 mg) of cosmetic sample is put into a 25mL colorimetric tube with a plug, 5mL of tetrahydrofuran is added, vortex oscillation is carried out for 1min for dispersion, methanol is added to fix the volume to scale, ultrasonic extraction is carried out for 15min, and partial liquid is filtered through a 0.22 mu m filter membrane, thus obtaining the sample solution No. 1.
2. UV measurement
The 7 single reference solutions of example 1 were taken and measured by full scanning at a wavelength range of 200-800nm using ultraviolet absorption spectroscopy. Detecting and obtaining the maximum absorption wavelength lambda max=325 nm of retinol; the maximum absorption wavelength λmax=380 nm of retinaldehyde; the maximum absorption wavelength λmax=325 nm of retinol acetate; maximum absorption wavelength λmax=325 nm of retinol propionate; the maximum absorption wavelength of retinol retinoic acid ester λmax=340 nm; maximum absorption wavelength λmax=325 nm of retinol palmitate; the maximum absorption wavelength λmax=360 nm of the hydroxy pinacolone retinoic acid ester.
3. HPLC determination
The sample solution 1# and a series of mixed reference solutions with different concentrations in example 1 are respectively detected by High Performance Liquid Chromatography (HPLC), the maximum absorption wavelength measured by UV is taken as the detection wavelength, the retention time is compared for qualitative determination, and the external standard method is adopted for quantitative determination. The HPLC detection is carried out on a series of mixed reference substance solutions with different concentrations respectively to obtain the linear relation between the chromatographic peak areas of retinol and 6 retinol derivatives and the contents of the corresponding retinol and 6 retinol derivatives, the corresponding standard working curves are drawn, and the regression equation of the standard working curves of the retinol and 6 retinol derivatives is calculated. And then carrying out HPLC detection on the sample solution 1# and substituting the chromatographic peak areas of the obtained retinol and 6 retinol derivatives into a regression equation of a standard working curve of the corresponding retinol and 6 retinol derivatives, thus obtaining 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 Agilent Eclipse Plus C chromatographic column (column length of 250mm, inner diameter of 4.6mm, particle size of 5 μm); retinol, retinol acetate, retinol propionate, retinol palmitate detection wavelength is 325nm, retinol retinoic acid ester detection wavelength is 340nm, hydroxy pinacolone retinoic acid ester detection wavelength is 360nm, and retinol detection wavelength is 380nm; the column temperature is 30 ℃; the flow rate is 1.0ml/min; the sample injection amount is 10 μl; the mobile phase is acetonitrile-tetrahydrofuran, wherein the A phase is acetonitrile and the B phase is tetrahydrofuran; the analysis time is 30min; gradient elution.
The specific procedure of 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;
12-23 min, phase A: the volume ratio of the phase B is 70:30-70:30;
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 solutions
1g (accurate to 0.1 mg) of cosmetic sample is put into a 25mL colorimetric tube with a plug, 4mL of tetrahydrofuran is added, vortex oscillation is carried out for 1.5min for dispersion, methanol is added to fix the volume to scale, ultrasonic extraction is carried out for 12min, and partial liquid is filtered through a 0.22 mu m filter membrane, thus obtaining the sample solution No. 2.
2. UV measurement
The determination of the maximum absorption wavelength of retinol and 6 retinol derivatives was the same 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 are respectively detected by High Performance Liquid Chromatography (HPLC), the maximum absorption wavelength measured by UV is taken as the detection wavelength, the retention time is compared for qualitative determination, and the external standard method is adopted for quantitative determination. The HPLC detection is carried out on a series of mixed reference substance solutions with different concentrations respectively to obtain the linear relation between the chromatographic peak areas of retinol and 6 retinol derivatives and the contents of the corresponding retinol and 6 retinol derivatives, the corresponding standard working curves are drawn, and the regression equation of the standard working curves of the retinol and 6 retinol derivatives is calculated. And performing HPLC detection on the sample solution 2# and substituting the chromatographic peak areas of the obtained retinol and 6 retinol derivatives into a regression equation of a standard working curve of the corresponding retinol and 6 retinol derivatives, so as to calculate 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 Agilent Eclipse Plus C chromatographic column (column length of 250mm, inner diameter of 4.6mm, particle size of 5 μm); retinol, retinol acetate, retinol propionate, retinol palmitate detection wavelength is 325nm, retinol retinoic acid ester detection wavelength is 340nm, hydroxy pinacolone retinoic acid ester detection wavelength is 360nm, and retinol detection wavelength is 380nm; the column temperature is 30 ℃; the flow rate is 1.0ml/min; the sample injection amount is 10 μl; the mobile phase is methanol-acetonitrile-tetrahydrofuran, wherein the A phase is methanol, the B phase is acetonitrile, and the C phase is tetrahydrofuran; the analysis time is 35min; gradient elution.
The specific procedure of gradient elution is:
0-14min, phase A: and B phase: the volume ratio of the phase C is 10:90:0-10:90:0;
14-16min, phase A: and B phase: the volume ratio of the phase C is 10:90:0-10:60:30;
16-27min, phase A: and B phase: the volume ratio of the phase C is 10:60:30-10:60:30;
27-29min, phase A: and B phase: the volume ratio of the phase C is 10:60:30-10:90:0;
29-35min, phase A: and B phase: the volume ratio of the phase C is 10:90:0-10:90:0.
Example 4
A series of mixed reference solutions of different concentrations were prepared as in example 1, and the concentrations of the components in the reference solutions are shown in Table 2. According to the HPLC conditions of step 3 in example 2, 10. Mu.l of the mixed reference solution was precisely sucked into a liquid chromatograph, the mixed reference concentration (. Mu.g/ml) was plotted on the abscissa, the peak area was plotted on the ordinate, and the standard regression equation, correlation coefficient and linear range of retinol and 6 retinol derivatives were measured and calculated, and the specific results are shown in Table 3. As is clear from Table 3, the retinol and 6 retinol derivatives have good linear relationship in the examined concentration range.
TABLE 3 Table 3
| Compounds of formula (I) | Linear equation | Correlation coefficient R 2 | Linear range (μg mL) -1 ) |
| Retinol | y＝29.561x-14.029 | 0.9997 | 5-80 |
| Retinaldehyde | y＝75.307x-38.208 | 1 | 10-160 |
| Retinol acetate | y＝18.118x-10.934 | 0.9998 | 10-160 |
| Retinol propionate | y＝71.561x-26.664 | 0.9998 | 5-80 |
| Retinol retinoic acid ester | 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 reference solution was diluted, and the mixture was measured under the HPLC conditions of step 3 in example 2, and the noise triple signal was taken as the detection limit to obtain retinol 1.0. Mu.g.mL -1 1.0 mug.mL of retinaldehyde -1 Retinol acetate 1.0 μg.ml -1 Retinol propionate 0.5. Mu.g.mL -1 Retinol retinoic acid ester 2.0 μg mL -1 Retinol palmitate 2.0. Mu.g.mL -1 Hydroxy pinacolone retinoate 1.0 μg mL -1 The method comprises the steps of carrying out a first treatment on the surface of the Taking ten times of noise signal as quantitative limit to obtain retinol 3.0 μg.mL -1 3.0 mug.mL of retinaldehyde -1 Retinol acetate 3.0 μg.ml -1 Retinol propionate 1.5. Mu.g.mL -1 Retinol retinoic acid ester 6.0 μg.ml -1 Retinol palmitate 6.0. Mu.g.mL -1 Hydroxy pinacolone retinoate 3.0 μg mL -1 . The specific data are shown in Table 4.
TABLE 4 Table 4
| Component name | Detection limit (μg/mL) -1 ) | Limit of quantification (μg mL) -1 ) |
| Retinol | 1.0 | 3.0 |
| Retinaldehyde | 1.0 | 3.0 |
| Retinol acetate | 1.0 | 3.0 |
| Retinol propionate | 0.5 | 1.5 |
| Retinol retinoic acid ester | 2.0 | 6.0 |
| Retinol palmitate | 2.0 | 6.0 |
| Hydroxy pinacolone retinoic acid ester | 1.0 | 3.0 |
Example 5
7 single reference solutions were prepared as in example 1, with 20. Mu.g.mL of retinol in the single reference solution -1 40 mug.mL of retinaldehyde -1 40. Mu.g.mL of retinol acetate -1 20 mug/mL of retinol propionate -1 Retinol retinoic acid ester 40 μg mL -1 40. Mu.g.mL of retinol palmitate -1 40. Mu.g.mL of hydroxy pinacolone retinoate -1 7 test solutions were prepared as in example 2, step 1. According to the HPLC condition of step 3 in example 2, 10 μl of the same sample solution is precisely sucked and injected into the liquid chromatograph, sample injection analysis is repeated 6 times, peak areas of each component are measured, and specific precision results are shown in Table 5. The results show that RSD for retinol and 6 retinol derivatives are retinol, respectively: 1.76%, retinaldehyde: 0.42%, retinol acetate: 0.77%, retinol propionate: 0.73%, retinol palmitate: 1.06%, retinol retinoic acid ester: 1.56%, hydroxy pinacolone retinoate: 0.42%. The chromatographic peak areas RSD are less than 2%, which indicates that the precision of the instrument is good.
TABLE 5
Example 6
Select essence without 7 retinol and its derivatives (blue beauty and crystal repair essence produced by UK cosmetic (Wuhan Co., ltd.)]Emulsion [ Mei Qin Lian Ying run-high-strength emulsion of Uo lan produced by Uo Yisi cosmetic (Wuhan Co., ltd.)]Cream [ blue beauty lotus moisturizing and strengthening face cream produced by Uo Yisi beauty product (Wuhan Co., ltd.)]3 kinds of cosmetics with different matrixes, each of which is 2 parts, 1g (accurate to 0.0001 g) of each part is taken, the mixed standard stock solution in the example 1 is respectively added into a 10mL colorimetric tube with a plug, and a methanol solution containing tetrahydrofuran with the volume ratio of 2:23 is added to fix the volume to a scale to prepare the cosmetic with retinol of 10 mug.mL -1 40 mug.mL of retinaldehyde -1 40. Mu.g.mL of retinol acetate -1 40. Mu.g.mL of retinol propionate -1 Retinol retinoic acid ester 40 μg mL -1 Retinol palmitate 20. Mu.g.mL -1 40. Mu.g.mL of hydroxy pinacolone retinoate -1 A test solution was prepared as in step 1 of example 2. 6 parts of the same batch of sample solution are taken, 10 μl of the sample solution is precisely sucked and injected into a liquid chromatograph, and the sample is respectively injected and analyzed according to the HPLC condition of the step 3 in the example 2, and the peak area is measured, and the specific repeatability result is shown in Table 6.
The results show that RSD for retinol and 6 retinol derivatives are retinol, respectively: 2.24%, retinaldehyde: 1.42%; 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%. The chromatographic peak areas RSD are all less than 3%, indicating that the method is good in repeatability.
TABLE 6
Example 7
Selecting essence without 7 types of retinol and derivatives thereof [ Meilin moisturizing and repairing essence of the fruit blue produced by Uygur cosmetology (Wuhan) Co., ltd ], emulsion [ Meilin moisturizing and toughening emulsion of the fruit blue produced by Uygur cosmetology (Wuhan) Co., ltd ], cream [ Meilin moisturizing and toughening cream of the fruit blue produced by Uygur cosmetology (Wuhan) Co., ltd ],3 types of cosmetics with different matrixes, 9 parts of each cosmetic, 1g (accurate to 0.0001 g) per part, respectively adding 0.25mL/0.5mL/1.0mL of mixed standard stock solution in example 1, 3 samples of three groups of mixed standard stock solutions, 3mL of tetrahydrofuran, 1min of dispersion, and methanol constant volume to scale, and performing ultrasonic extraction for 15min, so that the theoretical addition of the samples in 3 concentrations of retinol and retinol propionic acid are recovered in a sample by vortex
Esters 5. Mu.g.mL each -1 、10μg·mL -1 、20μg·mL -1 Retinaldehyde, retinyl acetate, retinyl retinoate, retinylAlcohol palmitate and hydroxy pinacolone retinoate are 10 mug.mL respectively -1 、20μg·mL -1 、40μg·mL -1 Part of the liquid was filtered through a 0.22 μm filter.
Then, 10. Mu.l of the sample was precisely sucked up and injected into a liquid chromatograph under the HPLC conditions of step 3 in example 2, and the results are shown in Table 7. As is clear from Table 7, the recovery rates of retinol and 6 retinol derivatives ranged from 92.37 to 112.0%. The results showed that the recovery of retinol and 6 derivatives was good at all three concentrations, low, medium and high, with a precision (RSD) of 0.20-6.14%.
RSD results show that the method has high accuracy. The test result is very similar to the actual addition concentration, and the test requirement can be met.
TABLE 7
In conclusion, the method for detecting the contents of retinol and 6 retinol derivatives in the cosmetics has the characteristics of simple pretreatment, reliable sample separation, accurate quantitative and qualitative properties, 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 above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (4)
1. A method for detecting the contents of retinol and 6 retinol derivatives in cosmetics comprises the following steps: adding a cosmetic sample into a first solvent for dispersion, then adding a second solvent for ultrasonic extraction, filtering, taking a test solution obtained from the filtrate, determining the detection wavelength by adopting an ultraviolet absorption spectrometry, and then carrying out qualitative and quantitative detection by adopting a high performance liquid chromatography to determine the contents of retinol and 6 retinol derivatives in the test solution;
wherein:
the ratio of the added mass of the cosmetic sample to the added volume of the first solvent is 1:1-10, g/mL,
the first solvent is tetrahydrofuran, and the second solvent is tetrahydrofuran,
the dispersion adopts vortex oscillation to disperse, the oscillation time of the vortex oscillation is 0.5-2.0min,
the second solvent is methanol, and the second solvent is methanol,
the ratio of the mass of the cosmetic sample added to the volume of the second solvent added is 1:15-24, g/mL,
the ultrasonic extraction time is 10-20min,
the filtering adopts a filter membrane for filtering;
the method adopts an ultraviolet absorption spectrometry to determine the detection wavelength, and then adopts a high performance liquid chromatography to carry out qualitative and quantitative detection, and comprises the following steps:
1) Adding a third solvent into the reference substances of the retinol and the 6 retinol derivatives for dissolution, and then fixing the volume to prepare mixed reference substance solutions of the retinol and the 6 retinol derivatives and 7 single reference substance solutions respectively;
2) Respectively detecting 7 single reference substance solutions in the step 1) by adopting an ultraviolet absorption spectrometry method, and determining the maximum absorption wavelength of retinol and 6 retinol derivatives;
3) Respectively detecting the sample solution and the mixed reference substance solution in the step 1) by adopting a high performance liquid chromatography, 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 retinol and 6 retinol derivatives in the sample solution by adopting an external standard method for quantitative determination;
in the step 1), the third solvent is a methanol solution containing tetrahydrofuran;
in 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 325-380nm; the mobile phase is selected from one of acetonitrile-tetrahydrofuran or methanol-acetonitrile-tetrahydrofuran, wherein when the mobile phase is acetonitrile-tetrahydrofuran, the A phase is acetonitrile, the B phase is tetrahydrofuran, and the analysis time is 30min; when the mobile phase is methanol-acetonitrile-tetrahydrofuran, the A phase is methanol, the B phase is acetonitrile, the C phase is tetrahydrofuran, and the analysis time is 35min; gradient elution;
when the mobile phase is acetonitrile-tetrahydrofuran, the specific procedure for 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;
12-23 min, phase A: the volume ratio of the phase B is 70:30-70:30;
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;
when the mobile phase is methanol-acetonitrile-tetrahydrofuran, the specific procedure for the gradient elution is:
0-14min, phase A: and B phase: the volume ratio of the phase C is 10:90:0-10:90:0;
14-16min, phase A: and B phase: the volume ratio of the phase C is 10:90:0-10:60:30;
16-27min, phase A: and B phase: the volume ratio of the phase C is 10:60:30-10:60:30;
27-29min, phase A: and B phase: the volume ratio of the phase C is 10:60:30-10:90:0;
29-35min, phase A: and B phase: the volume ratio of the phase C is 10:90:0-10:90:0;
the 6 retinol derivatives are respectively retinol, retinol acetate, retinol propionate, retinol retinoic acid ester, retinol palmitate and hydroxy pinacol retinoic acid ester.
2. The method for detecting the contents of retinol and 6 retinol derivatives in cosmetics according to claim 1, wherein the cosmetic sample is selected from one of essence, emulsion or cream.
3. The method for detecting the content of retinol and 6 retinol derivatives in cosmetics as defined in claim 1, wherein in step 2), the total scanning wavelength range of the ultraviolet absorption spectroscopy is 200-800nm.
4. The method for detecting the content of retinol and 6 retinol derivatives in cosmetics according to claim 1, wherein in step 3), the high performance liquid chromatography is performed by:
the column temperature is 25-35 ℃,
the flow rate is 0.8-1.2 ml/min,
the sample injection amount is 5-15 μl.
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