CN115616117B - High performance liquid chromatography determination method for seven natural antioxidants in cosmetics - Google Patents
High performance liquid chromatography determination method for seven natural antioxidants in cosmetics Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
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- G—PHYSICS
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
Abstract
The invention provides a high performance liquid chromatography determination method of seven natural antioxidants in cosmetics. The method comprises the following steps: preparation of standard stock solutions: accurately weighing salidroside, asiaticoside, quercetin, salvianolic acid B, rosmarinic acid, ferulic acid, and salicylic acid standard substances, and making into standard stock solution; preparation of mixed standard working solutions: mixing the standard substances according to a certain mass ratio to prepare a mixed standard working solution; preparing a sample solution to be tested: fully extracting natural antioxidant in a sample through vortex, ultrasonic and centrifugation; loading a sample on a machine: filtering the treated solution by a membrane and then measuring by high performance liquid chromatography; and (4) analyzing results: seven natural antioxidants were characterized by retention time and quantified by a standard curve. The method can be used for simultaneously detecting various natural antioxidants, is simple to operate, has high sensitivity, is suitable for detecting cosmetics in multiple batches, and provides technical support for product quality control in the cosmetic industry.
Description
Technical Field
The invention relates to the field of cosmetic analysis, in particular to a high performance liquid chromatography determination method for seven natural antioxidants in cosmetics.
Background
The skin is one of the largest organs of the human body and is also the first natural barrier protecting the body from external aggressions. However, with the lapse of time and aging, the human skin undergoes endogenous aging, which is closely related to genetics, and exogenous aging, which is mainly affected by the environment and the individual lifestyle. The atmospheric environment where people live depends on has a large amount of active oxygen free radicals, and the active oxygen free radicals continuously attack cell tissues of human bodies through strong oxidation reaction, interfere cell signal conduction, accelerate apoptosis and accelerate skin aging, and are particularly characterized in that the skin is dry, loose, crumbly, melanin is increased and even color spots appear, and the phenomena seriously troubles people who love beauty.
Most of the cosmetics sold on the market at present contain an antioxidant, and the antioxidant serving as an exogenous antioxidant can effectively eliminate oxygen free radicals, so that the skin achieves the effects of resisting aging, whitening, sun screening, moisturizing and the like. The antioxidants are various in variety and source, and people tend to select natural antioxidants, and natural antioxidants derived from plant extracts are more easily favored by consumers. At present, a plurality of plants including centella, rosemary, salvia miltiorrhiza, sea buckthorn, tea, olive, liquorice, centella, rose, clove and the like have been proved to have good antioxidant effect. The components in the plant with antioxidant effect mainly include phenolic acids, flavonoids, flavanols, and polysaccharides. The detection method of the antioxidant in the cosmetics generally adopts gas chromatography and liquid chromatography tandem mass spectrometry, but the application of the gas chromatography is limited by the polarity and boiling point of the compound, the liquid chromatography tandem mass spectrometry has expensive instruments and equipment, high detection cost and low popularization rate, the matrix effect of an oil sample is large, the quantification is not easy, the application of the method is limited, and the high performance liquid chromatography has high sensitivity on the antioxidant. The invention aims to construct a rapid, accurate and simple-to-operate high performance liquid chromatography determination method for seven natural antioxidants in cosmetics, which is applied to detection of multiple batches of cosmetics and provides technical support for product quality control in the cosmetics industry.
Disclosure of Invention
The technical problem to be solved is as follows: the invention aims to construct a rapid, accurate and simple-to-operate high performance liquid chromatography determination method for seven natural antioxidants in cosmetics, and apply the method to the detection of multiple batches of cosmetics to provide technical support for the product quality control of the cosmetic industry.
The technical scheme is as follows: a high performance liquid chromatography determination method for seven natural antioxidants in cosmetics comprises the following steps:
(1) Preparation of standard stock solutions: accurately weighing 10mg of seven natural antioxidant standard substances respectively, placing the seven natural antioxidant standard substances into a 10mL brown volumetric flask, and performing constant volume by using chromatographic pure methanol to prepare a standard stock solution with the concentration of 1 mg/mL;
(2) Preparation of mixed standard working solutions: respectively and accurately weighing 10mg of each of seven natural antioxidant standard substances, placing the seven natural antioxidant standard substances into a 10mL brown volumetric flask, and preparing a mixed standard stock solution with the concentration of 1mg/mL by using chromatographic pure methanol to fix the volume. The mixed standard stock solutions were formulated with chromatographically pure methanol to give mixed standard working solutions with a concentration gradient of 0.05. Mu.g/mL, 0.25. Mu.g/mL, 1.25. Mu.g/mL, 6.25. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL.
(3) Preparing a sample solution to be tested: if the sample is aqueous liquid, adding chromatographic pure methanol, fixing the volume to 25mL, uniformly mixing, filtering by a membrane, and measuring the filtrate by high performance liquid chromatography; if the sample is a paste or powder solid, adding 25mL of chromatographic pure methanol, uniformly mixing by vortex, then carrying out ultrasonic extraction, centrifuging, taking an upper layer solution, filtering by using a membrane, and measuring the filtrate by using high performance liquid chromatography;
(4) Loading a sample on a machine: selecting an instrument as a high performance liquid chromatograph, a diode array detector, a reversed phase C18 column as a chromatographic column, controlling the column temperature to 35 ℃, acetonitrile as a mobile phase A, 0.1% phosphoric acid solution as a mobile phase B, and performing an elution gradient procedure: 0 to 5min,5 to 10% of A and 95 to 90% of B;5 to 10min,10 to 25% of A and 90 to 75% of B;10 to 15min,25 to 40% of A and 75 to 60% of B;15 to 25min,40 to 60% of A and 60 to 40% of B;25 to 45min,60 to 85 percent of A and 40 to 15 percent of B; the flow rate is 1.0mL/min; the sample injection amount is 10 mu L; detection wavelength: asiaticoside is 205nm; the content of salidroside, salvianolic acid B, rosmarinic acid and salicylic acid is 286nm; ferulic acid is 316nm; quercetin is 360nm.
(5) And (4) analyzing results: injecting the standard stock solution prepared in the step (1), the mixed standard working solution prepared in the step (2) and the filtrate in the step (3) into a high performance liquid chromatograph, performing gradient elution and detection under the condition of the step (4), determining the retention time of each natural antioxidant according to the peak-off time of the standard stock solution, drawing a standard curve according to the mass concentration (X, mg/L) corresponding to the peak area (Y) of each natural antioxidant in the mixed standard working solution, and obtaining a linear regression equation so as to determine the content of different natural antioxidants in the sample.
Further, the step (3) specifically comprises the following steps:
(a) If the sample is aqueous liquid, accurately weighing 1.0g of the sample, placing the sample in a 25mL brown volumetric flask, adding chromatographic pure methanol to a constant volume, uniformly mixing, and then filtering the solution by using a filter membrane, wherein the filtrate is measured by high performance liquid chromatography;
(b) And if the sample is a paste or powder solid, adding 25mL of chromatographic pure methanol, uniformly mixing for 5-10min in a vortex mode, then carrying out ultrasonic extraction for 5-10min, finally centrifuging for 10-15min at the temperature of 4 ℃ and under the condition of 8000-12000r/min, taking an upper layer solution for filtering, and measuring the filtrate by high performance liquid chromatography.
Further, the size of the filter membrane in the step (3) is 13mm × 0.22 μm.
Further, the model of the high performance liquid chromatograph in the step (4) is Aglient1260.
Further, the model of the reversed phase C18 column in the step (4) is Aglient Parashell EC-C18, the specification is 4.6mm multiplied by 250mm, and the particle size of the packed particles is 5 μm.
Further, the cosmetic includes aqueous liquids, creams and powdered solids.
Has the advantages that:
1. the method can be used for simultaneously detecting various natural antioxidants, the pretreatment operation is simple, the detection period is greatly shortened, and the detection method is quick and accurate and has high sensitivity;
2. the method can be used for detecting the natural antioxidant in multiple batches of cosmetics, quickly evaluating the antioxidant performance of the cosmetics, can be well applied to the actual detection of the cosmetics, and provides technical support for the product quality control of the cosmetic industry.
Drawings
FIG. 1 is a high performance liquid chromatography separation chart of a mixed standard solution of seven different natural antioxidants;
FIG. 2 is a high performance liquid chromatogram of an antioxidant component in an aqueous liquid sample;
FIG. 3 is a high performance liquid chromatogram of the antioxidant component in a paste sample.
Detailed Description
The invention will be further described with reference to the following figures and examples, which are illustrative of the invention and are not to be construed as limiting the invention:
example 1
1. Reagents and materials
1.1 Methanol, acetonitrile: and (4) carrying out chromatographic purification.
1.2 Phosphoric acid: and 4, high-grade purity.
1.3 Ultrapure water.
1.4 Natural antioxidant standards: salidroside, asiaticoside, quercetin, salvianolic acid B, rosmarinic acid, ferulic acid, and salicylic acid (purity 98%).
1.5 Preparation of standard solution of natural antioxidant: respectively and accurately weighing 10mg of seven natural antioxidant standard substances, placing the seven natural antioxidant standard substances into a 10mL brown volumetric flask, and fixing the volume by using chromatographic pure methanol to prepare a standard stock solution with the concentration of 1 mg/mL.
1.6 Preparation of mixed standard working solutions: respectively and accurately weighing 10mg of each of seven natural antioxidant standard substances, placing the seven natural antioxidant standard substances into a 10mL brown volumetric flask, and preparing a mixed standard stock solution with the concentration of 1mg/mL by using chromatographic pure methanol to fix the volume. The mixed standard stock solution was prepared with chromatographically pure methanol as mixed standard working solutions with concentration gradients of 0.05. Mu.g/mL, 0.25. Mu.g/mL, 1.25. Mu.g/mL, 6.25. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL.
1.7 And (3) filter membrane: the gauge was 13 mm. Times.0.22. Mu.m.
2. Instrument and apparatus
The detector of the high performance liquid chromatograph is a diode array detector, an analytical balance, a high-speed refrigerated centrifuge, a high-frequency numerical control ultrasonic cleaner and a multi-tube vortex oscillator.
3. High performance liquid chromatography conditions
3.1 And (3) chromatographic column: an Aglient Parashell EC-C18 column, 5 μm,4.6mm by 250mm;
3.2 Mobile phase: mobile phase a was acetonitrile, mobile phase B was 0.1% phosphoric acid solution, elution gradient procedure: 0 to 5min,5 to 10% of A and 95 to 90% of B;5 to 10min,10 to 25% A and 90 to 75% B;10 to 15min,25 to 40% of A and 75 to 60% of B;15 to 25min,40 to 60 percent of A and 60 to 40 percent of B;25 to 45min,60 to 85% A and 40 to 15% B;
3.3 Flow rate: 1.0 μ L/min;
3.4 Column temperature: 35 ℃;
3.5 Sample introduction amount: 10 mu L of the solution;
3.6 Detection wavelength: asiaticoside is 205nm; the content of salidroside, salvianolic acid B, rosmarinic acid and salicylic acid is 286nm; ferulic acid is 316nm; quercetin is 360nm.
4. Detection of standard solutions
And (3) carrying out gradient elution and detection on the prepared seven natural antioxidant standard stock solutions under the chromatographic conditions, and determining the peak time of each natural antioxidant to qualify the retention time. Gradient elution and detection are carried out on the prepared mixed standard working solution under the same conditions, seven different natural antioxidant mixed standard solution high performance liquid chromatography separation charts are obtained, and the result is shown in figure 1. Drawing a standard curve according to the mass concentration (X, mg/L) corresponding to the peak area (Y) of each natural antioxidant in the mixed standard working solution to obtain a linear regression equation, determining the content of different natural antioxidants in the sample, and calculating a detection limit (S/N = 3) and a quantification limit (S/N = 10), wherein the results are shown in Table 1;
TABLE 1 Linear equation, correlation coefficient, detection limit and quantitative limit table of seven natural antioxidants in cosmetics
5. Precision and recovery experiments
The recovery rate is measured by performing 3 levels of standard addition recovery experiments in the aqueous liquid cosmetic blank matrix, repeating the experiments 6 times at each addition level, and the results are shown in table 2, wherein the results show that the recovery rate of 7 antioxidants in the blank matrix sample at 3 addition levels is 87.1% -103%, and the relative standard deviation is 0.68% -3.89%. The method is proved to be in accordance with the analysis and detection of seven natural antioxidants in the cosmetics;
TABLE 2 recovery and relative standard deviation recovery Table for seven natural antioxidants in aqueous liquid cosmetics (n = 6)
Example 2
Determination of antioxidant composition in aqueous liquid sample
1. Reagents and materials
Methanol (chromatographically pure), acetonitrile (chromatographically pure), phosphoric acid (guaranteed reagent), a filter membrane (13 mm. Times.0.22 μm), and ultrapure water.
2. Instrument and apparatus
The detector of the high performance liquid chromatograph is a diode array detector, an analytical balance, a high-speed refrigerated centrifuge, a high-frequency numerical control ultrasonic cleaner and a multi-tube vortex oscillator.
3. High performance liquid chromatography conditions
3.1 And (3) chromatographic column: an Aglient Parashell EC-C18 column, 5 μm,4.6mm by 250mm;
3.2 Mobile phase: mobile phase a was acetonitrile, mobile phase B was 0.1% phosphoric acid solution, elution gradient procedure: 0 to 5min,5 to 10% of A and 95 to 90% of B;5 to 10min,10 to 25% A and 90 to 75% B;10 to 15min,25 to 40% of A and 75 to 60% of B;15 to 25min,40 to 60% of A and 60 to 40% of B;25 to 45min,60 to 85% A and 40 to 15% B;
3.3 Flow rate: 1.0 μ L/min;
3.4 Column temperature: 35 ℃;
3.5 Sample introduction amount: 10 mu L of the solution;
3.6 Detection wavelength: 360nm at 0-10min; 286nm for 10-15min; 15-25min is 316nm; 286nm for 25 to 30min and 205nm for 30 to 45min.
4. Sample assay
An aqueous liquid cosmetic is prepared by accurately weighing 1.0g (accurate to 0.0001 g) of sample, placing in 25mL brown volumetric flask, adding chromatographic pure methanol to constant volume, mixing well, filtering, and measuring under the above chromatographic conditions, wherein the result is shown in figure 2, and the sample contains antioxidants quercetin, salvianolic acid B and salicylic acid according to the retention time of seven antioxidants.
Example 3
Determination of antioxidant composition in paste sample
1. Reagents and materials
Methanol (chromatogram purity), acetonitrile (chromatogram purity), phosphoric acid (senior purity), filter membrane (13 mm multiplied by 0.22 μm), and ultrapure water.
2. Apparatus and device
The detector of the high performance liquid chromatograph is a diode array detector, an analytical balance, a high-speed refrigerated centrifuge, a high-frequency numerical control ultrasonic cleaner and a multi-tube vortex oscillator.
3. High performance liquid chromatography conditions
3.1 A chromatographic column: an Aglient Parashell EC-C18 column, 5 μm,4.6 mm. Times.250 mm;
3.2 Mobile phase: mobile phase a was acetonitrile, mobile phase B was 0.1% phosphoric acid solution, elution gradient program: 0 to 5min,5 to 10% of A and 95 to 90% of B;5 to 10min,10 to 25% A and 90 to 75% B;10 to 15min,25 to 40% of A and 75 to 60% of B;15 to 25min,40 to 60 percent of A and 60 to 40 percent of B;25 to 45min,60 to 85 percent of A and 40 to 15 percent of B;
3.3 Flow rate: 1.0 μ L/min;
3.4 Column temperature: 35 ℃;
3.5 Sample injection amount: 10 mu L of the solution;
3.6 Detection wavelength: 360nm at 0-10min; 286nm at 10-15min; 15-25min is 316nm; 286nm for 25 to 30min and 205nm for 30 to 45min.
4. Sample assay
A paste moisture cream is prepared by accurately weighing 1.0g (accurate to 0.0001 g) of a sample, adding 25mL of chromatographic pure methanol, vortex and mixing for 5min, then performing ultrasonic extraction for 5min, finally centrifuging for 10min at 4 ℃ and 10000r/min, centrifuging, taking an upper layer solution filtering membrane, and performing measurement under the chromatographic condition, wherein the result is shown in figure 3, and the sample contains antioxidant salvianolic acid B and ferulic acid according to the retention time of seven antioxidants.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (4)
1. A high performance liquid chromatography determination method for seven natural antioxidants in cosmetics is characterized in that the seven natural antioxidants comprise: salidroside, asiaticoside, quercetin, salvianolic acid B, rosmarinic acid, ferulic acid, and salicylic acid; the method specifically comprises the following steps:
(1) Preparation of standard stock solutions: accurately weighing 10mg of seven natural antioxidant standard substances respectively, placing the seven natural antioxidant standard substances into a 10mL brown volumetric flask, and performing constant volume by using chromatographic pure methanol to prepare a standard stock solution with the concentration of 1 mg/mL;
(2) Preparation of mixed standard working solutions: accurately weighing 10mg of each of seven natural antioxidant standard substances, placing the seven natural antioxidant standard substances into a 10mL volumetric flask, and preparing a mixed standard stock solution with the concentration of 1mg/mL by using chromatographic pure methanol to fix the volume; preparing the mixed standard stock solution into a mixed standard working solution with the concentration gradient of 0.05 mu g/mL, 0.25 mu g/mL, 1.25 mu g/mL, 6.25 mu g/mL, 12.5 mu g/mL and 25 mu g/mL by using chromatographic pure methanol;
(3) Preparing a sample solution to be tested: if the sample to be detected is aqueous liquid, adding chromatographic pure methanol, fixing the volume to 25mL, uniformly mixing, filtering by using a membrane, and measuring the filtrate by using high performance liquid chromatography; if the sample to be detected is a paste or powder solid, adding 25mL of chromatographic pure methanol, uniformly mixing by vortex, then carrying out ultrasonic extraction, centrifuging, taking an upper layer solution, filtering by using a membrane, and measuring the filtrate by using high performance liquid chromatography;
(4) And (3) loading a sample on a machine: selecting an instrument as a high performance liquid chromatograph, a diode array detector as a detector, filling an Aglient Parashell EC-C18 column with the specification of 4.6mm multiplied by 250mm and the particle size of 5 mu m, wherein the column temperature is 35 ℃, a mobile phase A is acetonitrile, a mobile phase B is 0.1% phosphoric acid solution, and an elution gradient program comprises the following steps: 0 to 5min,5 to 10% by weight of A and 95 to 90% by weight of B;5 to 10min,10 to 25% of the total weight of the crude oil, and 90 to 75% of the total weight of the crude oil; 10 to 15min,25 to 40% by weight of A and 75 to 60% by weight of B; 15-25min, 40% -60% of A and 60% -40% of B; 25-45min, 60-85% A and 40-15% B; the flow rate is 1.0mL/min; the sample injection amount is 10 mu L; detection wavelength: asiaticoside is 205nm; the content of salidroside, salvianolic acid B, rosmarinic acid and salicylic acid is 286nm; ferulic acid is 316nm; quercetin is 360nm;
(5) And (4) analyzing results: injecting the standard stock solution prepared in the step (1), the mixed standard working solution prepared in the step (2) and the filtrate in the step (3) into a high performance liquid chromatograph, performing gradient elution and detection under the condition of the step (4), determining the retention time of each natural antioxidant according to the peak emergence time of the standard stock solution, drawing a standard curve according to the mass concentration corresponding to the peak area of each natural antioxidant in the mixed standard working solution, and obtaining a linear regression equation so as to determine the content of different natural antioxidants in the sample.
2. The high performance liquid chromatography determination method for seven natural antioxidants in a cosmetic according to claim 1, wherein the step (3) specifically comprises the following steps:
(a) If the sample is an aqueous liquid, accurately weighing 1.0g of the sample, placing the sample in a 25mL brown volumetric flask, adding chromatographically pure methanol to a constant volume, uniformly mixing, and then filtering the solution to obtain a filter membrane, wherein the filtrate is measured by high performance liquid chromatography;
(b) If the sample is a paste or powder solid, adding 25mL of chromatographic pure methanol, uniformly mixing for 5-10 min by vortex, then carrying out ultrasonic extraction for 5-10 min, finally centrifuging for 10-15 min at 4 ℃ under the condition of 8000-12000 r/min, taking an upper layer solution for filtering, and measuring the filtrate by high performance liquid chromatography.
3. The high performance liquid chromatography determination method of seven natural antioxidants in a cosmetic according to claim 1, characterized by comprising: in the step (3), the specification of the filter membrane is 13mm multiplied by 0.22 μm.
4. The high performance liquid chromatography assay method for seven natural antioxidants in a cosmetic according to claim 1, wherein: the model of the high performance liquid chromatograph in the step (4) is Aglient1260.
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