CN115219613A

CN115219613A - Method for detecting azelaic acid and potassium azeloyl diglycolate in cosmetics

Info

Publication number: CN115219613A
Application number: CN202210682555.3A
Authority: CN
Inventors: 许勇; 韩晶; 郑荣; 彭兴盛
Original assignee: SHANGHAI INSTITUTE FOR FOOD AND DRUG CONTROL
Current assignee: SHANGHAI INSTITUTE FOR FOOD AND DRUG CONTROL
Priority date: 2022-06-16
Filing date: 2022-06-16
Publication date: 2022-10-21
Anticipated expiration: 2042-06-16
Also published as: CN115219613B

Abstract

The invention discloses a method for simultaneously detecting azelaic acid and potassium azeloyl diglycinate in cosmetics. By adopting the method, the sample can be directly measured by a high performance liquid chromatograph without derivatization after being extracted by an extraction solvent, and the method is simple and convenient, easy to operate, strong in universality and good in reproducibility, and is more suitable for popularization and application of the method among laboratories.

Description

Method for detecting azelaic acid and potassium azeloyl diglycolate in cosmetics

Technical Field

The invention relates to the field of cosmetic detection and analysis, in particular to a method for detecting azelaic acid and potassium azeloyl diglycinate in cosmetics.

Background

Azelaic acid drugs were first marketed in France in 1989 as a new anti-acne drug, developed by Schering AG, germany. Topical azelaic acid pharmaceutical formulations currently exist in class 3: gels, creams and mucilages. In recent years, azelaic acid has a remarkable effect in the protection of the skin and the treatment of skin diseases, such as acne treatment, skin aging improvement, skin inflammation improvement, skin tumor improvement, and the like. It is effective in inhibiting acne bacillus and blocking fatty acid production on skin, and can also slow down keratinocyte growth, block melanin formation, prevent spot formation, and reduce melanin pigmentation. In recent years, azelaic acid is widely used in whitening and acne-removing cosmetics due to the effects of azelaic acid in whitening and spot removal.

Potassium azelate is a novel azelaic acid derivative which solves many formulation difficulties of azelaic acid, has better water solubility than azelaic acid, has the characteristics of small dosage and strong anti-inflammatory effect, and is therefore favored as an alternative product to azelaic acid in recent years by cosmetic manufacturers.

At present, in the 2015 edition of 'cosmetic hygiene Specifications' in China, no detection method and limit value regulation exist for azelaic acid and potassium azeloyl diglycinate, and the national standard plan is numbered as 201909-48-T-469 in the national standard plan: the detection of azelaic acid in cosmetics, namely gas chromatography, is under examination, but the method is a gas chromatography determination after derivatization, is relatively complicated and is not easy to operate. The method is not suitable for measuring azelaic acid in cosmetics with complicated matrixes and is easy to generate matrix interference. The method only measures azelaic acid in the cosmetics, and the content measuring method of potassium azeloyl diglycinate is not carried out. The literature reports about the method for measuring the content of azelaic acid at home and abroad mainly focus on the fields of medicines, biological products and the like, and the adopted analysis technologies mainly comprise high performance liquid chromatography, gas chromatography, capillary electrophoresis chromatography and the like. In the method, azelaic acid is required to be introduced for analysis after derivatization, so the operation steps are complicated and the reproducibility is poor. At present, no literature report exists on a method for measuring the content of potassium azeloyl diglycinate in cosmetics.

Disclosure of Invention

The invention aims to provide a method for simultaneously detecting azelaic acid and potassium azeloyldiglycinate in cosmetics aiming at the defects in the prior art, and the method is simple, convenient, easy to operate, strong in universality and good in reproducibility, and is suitable for popularization and application of methods in laboratories.

In order to achieve the purpose, the invention adopts the technical scheme that:

provides a method for detecting azelaic acid and potassium azeloyl diglycinate in cosmetics, which comprises the following steps:

step one, preparing a standard series of solutions: accurately weighing standard substances of azelaic acid and potassium azeloyl diglycinate respectively in a measuring flask, dissolving the standard substances with methanol, fixing the volume to scale, shaking up the standard substances to serve as standard stock solutions of azelaic acid and potassium azeloyl diglycinate, accurately transferring the standard stock solutions, fixing the volume with methanol, and preparing to obtain standard series solutions;

step two, preparing a solution to be detected: precisely weighing a sample to be detected, adding a sodium hydroxide aqueous solution-methanol solution, performing vortex treatment, adding the sodium hydroxide aqueous solution-methanol solution for constant volume, performing ultrasonic treatment at 50-60 ℃ for 60-90min, and centrifuging for 5-20min to obtain a solution to be detected;

and step three, connecting the standard series solution prepared in the step one and the solution to be detected prepared in the step two to a high performance liquid chromatography system for detection.

Further, the concentrations of the standard series of solutions are respectively: 10.0. Mu.g/mL, 50.0. Mu.g/mL, 100.0. Mu.g/mL, 200.0. Mu.g/mL, 500. Mu.g/mL.

Further, the specific preparation method of the solution to be tested comprises the following steps: weighing 0.20g of a sample to be detected, placing the sample in a 50mL colorimetric tube with a plug, adding 45mL of a sodium hydroxide aqueous solution-methanol solution, carrying out vortex treatment for 1min, adding the sodium hydroxide aqueous solution-methanol solution to constant volume of 50mL, carrying out ultrasonic treatment at 60 ℃ for 90min, and centrifuging at 10000rpm for 5min to obtain a solution to be detected.

Further, the high performance liquid chromatography conditions:

and (3) chromatographic column: B362V1 Swedish SVEAC8 column, 250mm × 4.6mm × 5 μm; or an equivalent chromatographic column;

column temperature: 30 ℃;

mobile phase: the solution A is phosphate buffer solution; the solution B is acetonitrile; gradient elution;

flow rate: 1.00mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 210nm.

Further, the gradient elution procedure was:

further, the preparation method of the phosphate buffer solution comprises the following steps: precisely weighing 2.0-2.5g of monopotassium phosphate, adding 1000mL of ultrapure water for dissolving, uniformly mixing, adding phosphoric acid for adjusting the pH value to 3.1, and performing suction filtration through a 0.45-micron filter membrane to obtain the potassium phosphate.

Further, the mass concentration of the phosphoric acid is 85%.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

1) The extraction method of the invention can rapidly, completely and accurately extract azelaic acid and potassium azeloyl diglycinate in cosmetics; the method for simultaneously determining azelaic acid and potassium azelate derivative in cosmetics by adopting the high performance liquid chromatography is simple, convenient, high-efficiency and easy to implement. By adopting the method, the sample can be directly measured by a high performance liquid chromatograph without derivatization after being extracted by an extraction solvent, and the method is simple and convenient, easy to operate, strong in universality and good in reproducibility, and is more suitable for popularization and application of the method among laboratories.

2) By adopting the detection method, the recovery rate of the azelaic acid and the potassium azeloyl diglycinate in the cosmetic sample can meet the requirement.

3) The detection method is adopted to detect the cosmetic sample actually containing azelaic acid and potassium azelate diglycinate, and the detection result is consistent with the actual content of the sample; the method is proved to be accurate in measurement result in practical application.

4) The detection method can greatly make up the blank of the method for preparing the azelaic acid and the potassium azeloyl diglycolate in the existing cosmetics, the determination result is close to the true value, and the detection capability is correspondingly improved, so the method has wide application prospect and good market potential.

Drawings

FIG. 1 is a high performance liquid chromatogram of a standard solution of azelaic acid and potassium azelate diglycinate in one embodiment of the present invention; wherein, 1-azelaic acid, 2-azelaic acid potassium diglycinate;

FIG. 2 is a high performance liquid chromatogram of a cosmetic sample solution containing azelaic acid in accordance with an embodiment of the present invention;

FIG. 3 is a high performance liquid chromatogram of a cosmetic sample solution containing potassium azeloyl diglycinate in an embodiment of the invention.

Detailed Description

The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The invention provides a method for detecting azelaic acid and potassium azeloyl diglycinate in cosmetics, which comprises the following steps:

1. reagents and materials

Except for other provisions, the reagents used in the invention are analytically pure or below, and the water is first-grade water specified in GB/T6682.

1.1 Standard substance, see Table A.

Table a standard substance information table

1.2 acetonitrile, chromatographic purity.

1.3 methanol, pure by chromatography.

1.4 phosphoric acid (85%), super pure.

1.5 sodium hydroxide, analytical purity

1.6 Potassium dihydrogen phosphate, analytically pure

1.7 aqueous sodium hydroxide solution (60 mmol/L): weighing 2.4g of sodium hydroxide (1.5), adding 1000mL of water, dissolving and uniformly mixing.

1.8 aqueous sodium hydroxide (60 mmol/L) -methanol solution (1+1): 100mL of aqueous sodium hydroxide solution (60 mmol/L) (1.7) was added with 100mL of methanol (1.3), and the mixture was mixed well.

1.9 phosphate buffer solution: precisely weighing 2.0401g of monopotassium phosphate (1.6), adding 1000mL of ultrapure water for dissolving, uniformly mixing, adding phosphoric acid (1.4) for adjusting the pH value to 3.1, and performing suction filtration through a 0.45-micrometer filter membrane to obtain the potassium dihydrogen phosphate.

1.10 standard stock solutions azelaic acid and potassium azeloyldiglycinate standard stock solutions (ρ =1 g/L): respectively and precisely weighing 0.01g (precisely to 0.0001 g) of standard substances (1.1) of azelaic acid and potassium azeloyl diglycinate into a 10mL measuring flask, dissolving the standard substances with methanol (1.3), fixing the volume to a scale, and shaking up to obtain standard stock solutions of azelaic acid and potassium azeloyl diglycinate. Storing in a refrigerator at-18 deg.C in dark.

1.11 Standard series solutions 0.10mL, 0.50mL, 1.00mL, 2.00mL, 5.00mL, to 10mL measuring bottles were accurately removed, and the volume to the scale was determined with methanol (1.3), shaken, and prepared into standard series solutions with concentration of 10.0. Mu.g/mL, 50.0. Mu.g/mL, 100.0. Mu.g/mL, 200.0. Mu.g/mL, 500. Mu.g/mL.

2. Apparatus and device

2.1 high performance liquid chromatograph, ultraviolet detector.

2.2 analytical balance: sensory amounts 0.0001g and 0.00001.

2.3 high-speed centrifuger (the rotating speed is more than or equal to 10000 rpm).

2.4 vortex mixer.

2.5 ultrasonic cleaner

3. Sample processing

Weighing 0.20g (accurate to 0.001 g) of a sample, placing the sample in a 50mL colorimetric tube with a plug, adding 45mL of a sodium hydroxide aqueous solution (60 mmol/L) -methanol solution (1+1) (1.8), carrying out vortex treatment for 1min, adding another sodium hydroxide aqueous solution (60 mmol/L) -methanol solution (1+1) (1.8) to fix the volume to 50mL, carrying out ultrasonic treatment at 60 ℃ for 90min, and centrifuging at 10000rpm for 5min to obtain a solution to be detected.

4. Detection of

Connecting the sample solution containing azelaic acid and potassium azeloyl diglycinate and the standard series solution to a high performance liquid chromatography system for detection.

High performance liquid chromatography conditions:

a chromatographic column: B362V1 Swedish SVEAC8 column (250 mm. Times.4.6 mm. Times.5 μm), or equivalent chromatography column;

column temperature: 30 ℃;

mobile phase: solution A: phosphate buffer solution (1.9); solution B: acetonitrile (1.2), gradient elution procedure see table 1.

TABLE 1 gradient elution procedure

Flow rate: 1.00mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 210nm.

Example 1

Positive sample labeling assay: selecting three positive samples containing azelaic acid or potassium azelate, carrying out a positive standard adding test on the positive samples, adding a component to be detected which is equivalent to the samples, and determining the recovery rate result. Sample information is detailed in table 2.

TABLE 2 sample information

In example 1, the main instruments, materials and reagents used include:

1. instruments and reagents

Agilent1260 high performance liquid chromatograph (Agilent, usa); sartorius CP224S and 225D-1CN electronic balances (Sartorius, germany); model 5800 sonicator (Branson, usa); MS3 vortex mixer (IKA, germany); 5810R type bench centrifuge (eppendorf, germany); a Milli-Q Reference model A + ultrapure water meter (Millipore, USA).

Azelaic acid standard with purity of 99% (mass fraction, m/m, alatin); potassium nonanedioyl diglycoate standard, purity 58.812% (mass fraction, m/m, shanghai mclin biotechnology limited) acetonitrile, methanol (chromatograhy, merk, germany); phosphoric acid, sodium hydroxide, potassium dihydrogen phosphate (analytical purity, shanghai Ling Fenghua Chemicals Co., ltd.). The water is ultrapure water.

2. Chromatographic conditions

column temperature: 30 ℃;

mobile phase: solution A: a phosphate buffer solution; solution B: acetonitrile, gradient elution procedure see table 1.

TABLE 1 gradient elution procedure

Phosphate buffer solution: precisely weighing 2.0401g of potassium dihydrogen phosphate, adding 1000mL of ultrapure water for dissolving, mixing, adding phosphoric acid (1.4) for adjusting pH to 3.1, and filtering with 0.45 μm filter membrane.

Flow rate: 1.00mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 210nm.

Determination of linear relationship in HPLC system:

standard stock solutions of azelaic acid and potassium azeloyldiglycinate (ρ =1 g/L): respectively and precisely weighing 0.01g (precisely to 0.0001 g) of standard substances of azelaic acid and potassium azeloyl diglycinate in a 10mL measuring flask, dissolving the standard substances with methanol, fixing the volume to a scale, and shaking up to obtain standard stock solutions of azelaic acid and potassium azeloyl diglycinate. Storing in a refrigerator at-18 deg.C in dark.

The standard series solution is accurately transferred into a measuring flask with the standard stock solution of 0.10mL, 0.50mL, 1.00mL, 2.00mL, 5.00mL and 10mL, the volume is fixed to the scale by methanol, and the standard series solution with the concentration of 10.0 mug/mL, 50.0 mug/mL, 100.0 mug/mL, 200.0 mug/mL and 500 mug/mL is prepared by shaking up.

Linear regression analysis was performed using peak area as ordinate (y) and concentration as abscissa (x, μ g/mL) to obtain the linear equation shown in Table 3 below.

TABLE 3 Standard Curve for azelaic acid and potassium azelamide

4. And (3) treating a positive sample by adding a sample solution with a standard recovery rate: respectively taking 0.1g (accurate to 0.001 g) of each of the azelaic acid positive sample and the potassium azeloyl diglycinate positive sample, respectively and accurately adding azelaic acid or potassium azeloyl diglycinate standard substances with the amount equivalent to that in the samples, placing the samples into a 50mL colorimetric tube with a plug, adding 45mL of sodium hydroxide aqueous solution (60 mmol/L) -methanol solution (1+1)), carrying out vortex treatment for 1min, adding sodium hydroxide aqueous solution (60 mmol/L) -methanol solution (1+1) to reach 50mL, carrying out ultrasonic treatment for 90min at the temperature of 60 ℃, and then centrifuging at 10000rpm for 5min to obtain a solution to be detected. Test solutions were prepared as recovery solutions, and the recovery and relative standard deviation were calculated by repeating the experiment 6 times per standard addition level (n = 6).

Aqueous sodium hydroxide solution (60 mmol/L): weighing 2.4g of sodium hydroxide, adding 1000mL of water, dissolving and uniformly mixing.

Aqueous sodium hydroxide (60 mmol/L) -methanol solution (1+1): 100mL of aqueous sodium hydroxide solution (60 mmol/L) was added with 100mL of methanol (1.3), and the mixture was mixed well.

The calculation of azelaic acid and potassium azeloyl diglycinate in the sample is given by equation (1):

(1)

in the formula: omega-content of azelaic acid and potassium azeloyl diglycinate in the cosmetic,%;

m-sample size, g;

rho is substituted into the standard curve to calculate the mass concentration of the azelaic acid and the potassium azeloyl diglycinate in the cosmetics obtained by the standard curve, namely mu g/mL;

v-volume, mL.

Under the chromatographic condition, a standard working curve solution and a sample solution are respectively sampled and are characterized according to retention time and a spectrogram of a diode array. The contents of azelaic acid and potassium azeloyl diglycinate in the sample solution were determined from the standard curve. The response value of the azelaic acid and the potassium azeloyldiglycinate in the sample solution is in the linear range of the standard curve, and if the response value exceeds the linear range, the extracting solution is diluted, and the amount of the extracting solution is measured or increased for re-detection.

The results of the positive spiked recovery determinations are shown in table 4, which indicates that satisfactory results were obtained for the recovery determinations.

TABLE 4 Positive sample recovery and precision test results

Example 2

And (3) blank sample labeling determination: three blank matrix samples of gel, cream emulsion and water without azelaic acid and potassium azeloyl diglycolate are taken, blank standard adding tests are carried out on the samples, and the recovery rate result is measured.

In example 2, the main instruments, materials and reagents used included:

1. instruments and reagents

As in example 1 above.

2. Chromatographic conditions

As in example 1 above.

Linear relationship determination in HPLC systems

Accurately transferring the standard stock solution (1.10) into a measuring flask with the concentration of 0.10mL, 0.50mL, 1.00mL, 2.00mL, 5.00mL or 10mL, metering to a scale with methanol (1.3), shaking up, and preparing into standard series solutions with the concentrations of 10.0. Mu.g/mL, 50.0. Mu.g/mL, 100.0. Mu.g/mL, 200.0. Mu.g/mL and 500. Mu.g/mL.

Linear regression analysis was performed using peak area as ordinate (y) and concentration as abscissa (x, μ g/mL) to obtain the linear equation shown in Table 5 below.

TABLE 5 Standard Curve for azelaic acid and potassium azeloyl diglycinate

4. Processing a blank sample with standard recovery rate: weighing 0.20g (accurate to 0.001 g) of blank matrix samples of gel, cream emulsion and water respectively, placing the blank matrix samples into a 50mL colorimetric tube with a plug, precisely adding a certain amount of standard azelaic acid and potassium azelainate, adding 45mL of sodium hydroxide aqueous solution (60 mmol/L) -methanol solution (1+1)), performing vortex treatment for 1min, adding sodium hydroxide aqueous solution (60 mmol/L) -methanol solution (1+1) to fix the volume to 50mL, performing ultrasonic treatment at 60 ℃ for 90min, and centrifuging at 10000rpm for 5min to obtain a solution to be detected. The experiment was repeated 6 times per spiked level and the recovery and relative standard deviation (n = 6) were calculated.

(1)

m-sample size, g;

v is volume of constant volume, mL.

The results of the blank matrix standard recovery determination are shown in table 6, which indicates that the recovery determination gives satisfactory results.

TABLE 6 recovery and precision test results for blank matrix spiking

Example 3

Respectively extracting and detecting a cosmetic 1 which is produced by a certain plant A and added with azelaic acid and a cosmetic 2 which is produced by a certain plant B and added with potassium azeloyl diglycinate: the marked content of azelaic acid in the sample from plant A is 20%; and 5% of potassium azeloyl diglycinate in the sample of the B factory.

In example 3, the main instruments, materials and reagents used included:

1. instruments and reagents

As in example 1 above.

2. Chromatographic conditions

As in example 1 above.

Determination of linear relationship in HPLC system:

standard stock solutions of azelaic acid and potassium azeloyldiglycinate (ρ =1 g/L): respectively and precisely weighing 0.01g (precisely to 0.0001 g) of standard substances of azelaic acid and potassium azeloyl diglycinate in a 10mL measuring flask, dissolving the standard substances with methanol, fixing the volume to a scale, and shaking up to obtain standard stock solutions of azelaic acid and potassium azeloyl diglycinate. Storing in refrigerator at-18 deg.C in dark place.

Linear regression analysis was performed using peak area as ordinate (y) and concentration as abscissa (x, μ g/mL) to obtain the linear equation shown in Table 7 below.

TABLE 7 Linear relationship of azelaic acid and potassium azeloyl diglycinate

4. Sample processing

Weighing 0.20g (accurate to 0.001 g) of a sample, placing the sample in a 50mL colorimetric tube with a plug, adding 45mL of a sodium hydroxide aqueous solution (60 mmol/L) -methanol solution (1+1)), carrying out vortex treatment for 1min, adding a sodium hydroxide aqueous solution (60 mmol/L) -methanol solution (1+1) to the volume of 50mL, carrying out ultrasonic treatment at 60 ℃ for 90min, and centrifuging at 10000rpm for 5min to obtain a solution to be detected.

Aqueous sodium hydroxide (60 mmol/L) -methanol solution (1+1): 100mL of aqueous sodium hydroxide solution (60 mmol/L) was added with 100mL of methanol, and the mixture was mixed well.

(1)

m-sample size, g;

v is volume of constant volume, mL.

The sample measurement results are shown in table 8, and the extraction method and the rapid measurement method can accurately quantify the content of azelaic acid and potassium azeloyldiaglycinate in the sample.

TABLE 8 determination of azelaic acid in the samples

In conclusion, the method for detecting azelaic acid and potassium azeloyl diglycinate in the cosmetics is simple, convenient and quick to operate and high in sensitivity, and the recovery rate and the repeatability of the method meet the requirements of daily detection. The method has the advantages of high sensitivity, high quality and accuracy and feasible linear range, and greatly improves the monitoring capability of a detection mechanism on the cosmetics.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. A method for detecting azelaic acid and potassium azeloyl diglycinate in cosmetics is characterized by comprising the following steps:

2. The detection method according to claim 1, wherein the concentrations of the standard series of solutions are respectively: 10.0. Mu.g/mL, 50.0. Mu.g/mL, 100.0. Mu.g/mL, 200.0. Mu.g/mL, 500. Mu.g/mL.

3. The detection method according to claim 1, wherein the specific preparation method of the solution to be detected is as follows: weighing 0.20g of a sample to be detected, placing the sample in a 50mL colorimetric tube with a plug, adding 45mL of a sodium hydroxide aqueous solution-methanol solution, carrying out vortex treatment for 1min, adding the sodium hydroxide aqueous solution-methanol solution to constant volume of 50mL, carrying out ultrasonic treatment at 60 ℃ for 90min, and centrifuging at 10000rpm for 5min to obtain a solution to be detected.

4. The detection method according to claim 1, wherein the high performance liquid chromatography conditions are:

a chromatographic column: B362V1 Swedish SVEAC8 column, 250mm × 4.6mm × 5 μm; or an equivalent chromatographic column;

column temperature: 30 ℃;

flow rate: 1.00mL/min;

sample introduction amount: 10 mu L of the solution;

detection wavelength: 210nm.

5. The detection method according to claim 4, wherein the gradient elution procedure is:

6. the detection method according to claim 4, wherein the phosphate buffer solution is prepared by a method comprising: precisely weighing 2.0-2.5g of monopotassium phosphate, adding 1000mL of ultrapure water for dissolving, uniformly mixing, adding phosphoric acid for adjusting the pH value to 3.1, and performing suction filtration through a 0.45-micron filter membrane to obtain the potassium phosphate.

7. The detection method according to claim 6, wherein the phosphoric acid is at a mass concentration of 85%.