阅读说明：本技术 一种化妆品中玻色因的液相检测方法 (Liquid phase detection method for vitronectin in cosmetics ) 是由 王甜甜 饶亮明 李兴德 于 2020-12-28 设计创作，主要内容包括：本发明提供一种化妆品中玻色因的液相检测方法,属于化妆品检测领域。该检测方法为HPLC-示差折光检测法,HPLC的色谱条件为：色谱柱采用氨基柱,流动相为75～85％的乙腈,等度洗脱,检测器为示差折光检测器,进样量为5～12μL。该方法的样品前处理简单、检测灵敏度高,线性范围宽,能够实现对各种化妆品中玻色因的含量进行有效检测。(The invention provides a liquid phase detection method of a vitronectin in cosmetics, belonging to the field of cosmetic detection. The detection method is an HPLC-differential refraction detection method, and the chromatographic conditions of the HPLC are as follows: the chromatographic column adopts an amino column, the mobile phase is 75-85% of acetonitrile, isocratic elution is carried out, the detector is a differential refraction detector, and the sample injection amount is 5-12 mu L. The method has the advantages of simple sample pretreatment, high detection sensitivity and wide linear range, and can realize effective detection of the content of the vitreous chromogen in various cosmetics.)

1. A liquid phase detection method of vitronectin in cosmetics is characterized in that the method is an HPLC-differential refraction detection method, and the chromatographic conditions of the HPLC are as follows: an amino column is adopted as a chromatographic column, 75-85% of acetonitrile is used as a mobile phase, isocratic elution is carried out, a detector is a differential refraction detector, and the sample injection amount is 5-12 mu L;

the detection step comprises:

A. preparing a standard working curve: accurately weighing a vitreous color factor standard substance, preparing 25-50 mg/ml standard stock solution by using ultrapure water, diluting the stock solution into a series of vitreous color factor solutions with different concentrations in a doubling way, and obtaining a standard curve by integrating the response peak area with the concentration after detecting by adopting the chromatographic condition of HPLC;

B. sample preparation: dispersing a sample to be detected by trichloromethane, adding ultrapure water for vortex extraction, centrifuging for 8-15 min at 8000-15000 rmp/min, taking supernatant, and filtering filtrate for detection;

C. sample detection: and after detecting the sample to be detected by adopting the chromatographic condition of the HPLC, obtaining the response peak area of the vitreous color factor contained in the sample to be detected, substituting the response peak area into the standard curve for calculation, and obtaining the concentration of the vitreous color factor contained in the sample to be detected.

2. The liquid-phase detection method of vitronectin in cosmetics according to claim 1, wherein in the step of preparing the standard working curve, the concentrations of a series of vitronectin solutions are respectively as follows: 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 25 mg/mL.

3. The method for detecting a vitreous color factor in a cosmetic according to claim 1, wherein the chromatographic condition of HPLC is 65 to 72 ℃.

4. The method for detecting vitreous color in cosmetic according to claim 1, wherein the flow rate is 0.5 to 1mL/min under the chromatographic conditions of HPLC.

5. The liquid-phase detection method of vitronectin in cosmetics according to claim 1, wherein the sample preparation step is a step of filtering the supernatant using a 0.45 μm aqueous phase filter.

6. The method for detecting the liquid phase of the vitreous chromogen in the cosmetic according to claim 1, wherein the method for preparing the standard working curve is adopted, the obtained standard curve is y-0.7273 x, the correlation coefficient is 0.996, and the linear range is 5-25 mg/mL.

7. The method for liquid-phase detection of vitronectin in a cosmetic according to claim 1, wherein the detection limit is 0.2g/100g by the HPLC-differential refractometry.

8. The method for detecting a vitreous color in a cosmetic according to claim 1, wherein the amino column is Shodex Asahipak NH₂P-50 4E。

Technical Field

The invention relates to the field of cosmetic detection, in particular to a liquid phase detection method of a vitreous chromogen in cosmetics.

Background

Vitronectin (Pro-xylone), also known as hydroxypropyl tetrahydropyrane triol, is a bioactive substance and can promote the production of glycosaminoglycan (GAG) and further promote the production of proteoglycan, and meanwhile, vitronectin can promote the synthesis of certain cytokines and collagen through GAG in the process, so that vitronectin has the symptoms of resisting skin aging, dehydration and the like, and the skin becomes tight and elastic. In addition, some experiments prove that the vitronectin can promote the regeneration of fibroblast growth factor (FGF, similar to EGF) and can effectively repair damaged skin. Therefore, the glass color is widely applied to the field of cosmetics, and is mainly applied to high-end skin care products such as anti-wrinkle products.

At present, various cosmetics are on the market, all claim to contain the vitreous color cause, but few cosmetics can clearly indicate the content of the vitreous color cause. Even if the product indicates relevant concentration, the concentration of the active substance is only the concentration of the contained vitronectin, but not the concentration of pure vitronectin, such as the Helianna product in Eurya flag, the concentration of the active substance is generally 30%, but the actual concentration of the contained vitronectin is not published. Therefore, how to quickly and effectively detect the actual content of the vitreous color factor in various skin care products has a very important significance for the quality control of related products, and the existing detection method is difficult to realize the effective detection of the vitreous color factor content in the cosmetics.

In view of this, the present application provides a method for detecting a vitreous color factor.

Disclosure of Invention

The invention aims to provide a liquid-phase detection method of the vitronectin in cosmetics, which has the advantages of simple sample pretreatment, high detection sensitivity and wide linear range and can realize effective detection of the content of the vitronectin in various cosmetics.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a liquid phase detection method of vitronectin in cosmetics is an HPLC-differential refraction detection method, and the chromatographic conditions of the HPLC are as follows: an amino column is adopted as a chromatographic column, 75-85% of acetonitrile is used as a mobile phase, isocratic elution is carried out, a detector is a differential refraction detector, and the sample injection amount is 5-12 mu L;

the detection step comprises:

A. preparing a standard working curve: accurately weighing a vitreous color factor standard substance, preparing 25-50 mg/ml standard stock solution by using ultrapure water, diluting the stock solution into a series of vitreous color factor solutions with different concentrations in a doubling way, and obtaining a standard curve by integrating the response peak area with the concentration after detecting by adopting the chromatographic condition of HPLC;

B. sample preparation: dispersing a sample to be detected by trichloromethane, adding ultrapure water for vortex extraction, centrifuging for 8-15 min at 8000-15000 rmp/min, taking supernatant, and filtering filtrate for detection;

C. sample detection: and after detecting the sample to be detected by adopting the chromatographic condition of the HPLC, obtaining the response peak area of the vitreous color factor contained in the sample to be detected, substituting the response peak area into the standard curve for calculation, and obtaining the concentration of the vitreous color factor contained in the sample to be detected.

Further, in a preferred embodiment of the present invention, in the step of preparing the standard working curve, the concentrations of a series of the vitronectin solutions are: 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 25 mg/mL.

Further, in a preferred embodiment of the present invention, the chromatographic conditions of the HPLC are 65-72 ℃.

Further, in a preferred embodiment of the present invention, the flow rate of the HPLC chromatogram is 0.5-1 mL/min.

Further, in a preferred embodiment of the present invention, in the sample preparation step, the supernatant is filtered using a 0.45 μm aqueous phase filter.

Further, in a preferred embodiment of the present invention, the method for preparing the standard working curve is adopted, and the obtained standard curve is y-0.7273 x, the correlation coefficient is 0.996, and the linear range is 5-25 mg/mL.

Further, in a preferred embodiment of the present invention, the detection limit is 0.2g/100g using the HPLC-refractometry method.

Further, in the preferred embodiment of the present invention, the amino column is Shodex Asahipak NH₂P-50 4E。

The invention has the following effects:

the liquid-phase detection method for the vitreous color factor in the cosmetics provided by the invention adopts an HPLC-differential refraction detection method, has the advantages of strong specificity, high sensitivity, good accuracy, wide linear range and simple and rapid sample pretreatment process, can realize effective detection of the content of the vitreous color factor in various cosmetics, and is beneficial to the quality control of various cosmetics taking the vitreous color as a main active ingredient.

Drawings

FIG. 1 is a liquid chromatogram of a standard control in example 1 of the present invention;

FIG. 2 is a standard curve in example 1 of the present invention;

FIG. 3 is a liquid chromatogram of sample No. 1 to be tested in example 1 of the present invention;

fig. 4 is a liquid chromatogram of sample No. 2 to be tested in example 1 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

The embodiment provides a liquid phase detection method of vitronectin in cosmetics, which comprises the following steps:

(1) preparing a standard curve:

accurately weighing 50mg of a vitronectin standard substance, preparing a standard stock solution of 50mg/mL by using ultrapure water, diluting by times to 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL and 25mg/mL, detecting by using an HPLC-differential refraction detection method to obtain a chromatogram shown in figure 1, wherein a chromatographic peak with retention time at 5.4min is vitronectin, and integrating the concentration by a response peak area to obtain a standard curve shown in figure 2: y is 0.7273x, and the correlation coefficient is 0.9963;

wherein, the chromatographic conditions of HPLC are as follows: the chromatographic column adopts Shodex Asahipak NH₂P-504E, wherein the mobile phase is 80% acetonitrile, isocratic elution is carried out, the detector is a differential refraction detector, the sample injection amount is 10 mu L, the column temperature is 68 ℃, and the flow rate is 0.8 mL/min;

(2) sample preparation:

no. 1 sample to be tested is traceless anti-wrinkle cream sold in a certain company.

Weighing 1g of sample to be tested, adding 5ml of trichloromethane for dispersion, adding 5ml of ultrapure water for vortex extraction, centrifuging for 10min at 10000rmp/min, taking supernatant, and filtering by using a 0.45 mu m water-phase filter membrane to obtain No. 1 sample test solution to be tested.

(3) Sample detection:

the sample test solution of sample No. 1 was detected under the chromatographic conditions of the HPLC in the step (1) to obtain a chromatogram shown in fig. 3, and the response peak area of the vitreous color factor in the chromatogram was calculated by substituting the standard curve y of 0.7273x to obtain a concentration of the vitreous color factor contained in the sample of 59 mg/g.

Example 2

The embodiment provides a liquid phase detection method of vitronectin in cosmetics, which comprises the following steps:

(2) preparing a standard curve:

accurately weighing 50mg of a vitreous chromogen standard substance, preparing 50mg/mL standard stock solution by using ultrapure water, diluting by times to 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL and 25mg/mL, detecting by using an HPLC-differential refraction detection method, and integrating the concentration by using a response peak area to obtain a standard curve: y is 0.7273x, and the correlation coefficient is 0.9963;

wherein, the chromatographic conditions of HPLC are as follows: the chromatographic column adopts Shodex Asahipak NH 2P-504E, the mobile phase is 75% acetonitrile, isocratic elution is carried out, the detector is a differential refraction detector, the sample injection amount is 5 mu L, the column temperature is 72 ℃, and the flow rate is 0.5 mL/min;

(2) sample preparation:

the vitronectin raw material obtained by a chemical synthesis method is a No. 2 sample to be detected, and the components contained in the vitronectin raw material are vitronectin and water. Filtering with 0.45 μm water-phase filter membrane to obtain No. 2 sample to be tested.

(3) Sample detection:

the sample test solution of sample No. 2 was detected under the chromatographic conditions of the HPLC in the step (1) to obtain a chromatogram shown in fig. 4, and the response peak area of the vitreous color factor in the chromatogram was calculated by substituting the standard curve y of 0.7273x to obtain a concentration of the vitreous color factor contained in the sample of test of 48.05%.

The liquid phase detection method of the vitronectin provided by the invention is examined by the methodology through the following experiments, and the results are as follows:

1. and (3) repeatability inspection:

the same batch of vitreous chromogen raw materials obtained by a chemical synthesis method is taken as a sample, a test solution is prepared, the test method provided in the embodiment 1 is used for carrying out measurement for a plurality of times, and the calculation results are shown in the table 1:

TABLE 1 results of the repeatability tests

The results show that: the liquid phase detection method for vitronectin provided in example 1 is good in repeatability.

2. Precision survey

Taking a standard sample solution, continuously injecting samples for 6 times by using the detection method provided in example 1, wherein the sample injection amount is 10 mu L each time, measuring the peak area of each time, and the result is shown in Table 2:

TABLE 2 results of the precision test

The results show that: the liquid phase detection method of vitronectin provided in example 1 is excellent in accuracy.

3. Intermediate precision survey:

the content of vitreous color in the same product (MODE ball traceless anti-wrinkle cream) was determined by two analysts using the detection method provided in example 1, twice per person, and averaged, with the results shown in table 3:

TABLE 3 results of intermediate precision investigation

The results show that: the results of two analysts measuring the content of the vitreous color factor in the same product are close, which shows that the intermediate precision of the liquid phase detection method of the vitreous color factor provided in example 1 is good.

4. And (4) inspecting the recovery rate:

the recovery rates of the detection methods provided in example 1 were examined using the spiked recovery method, and the results are shown in table 4:

TABLE 4 recovery test results

The results show that the detection method provided in example 1 has high recovery and high accuracy, indicating that the method is highly reliable.

5. Sensitivity investigation:

and analyzing by adopting a stepwise dilution method and using standard vitronectin solutions with different concentrations, and measuring that the lowest detection concentration of the vitronectin is 0.2g/100g under the condition that the signal to noise ratio is 3.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.