Skin saccharification degree evaluation method and skin saccharification degree detection device

Abstract

The invention provides a skin saccharification degree evaluation method and a skin saccharification degree detection device, by using the skin saccharification degree evaluation method and the skin saccharification degree detection device provided by the invention, the skin saccharification degree can be conveniently, quickly and accurately evaluated; according to the skin saccharification degree evaluation result obtained by the method or the detection device provided by the invention, a proper skin care product can be selected in a more targeted manner for skin care, and a better skin state can be obtained.

Description

Technical Field

[0001] This invention relates to a method for evaluating the degree of skin glycation and a device for detecting the degree of skin glycation. Background Technology

[0002] It is known that oxidation and glycation reactions in the body have adverse effects on cells and tissues, especially proteins that undergo oxidation or glycation, i.e., oxidized proteins or glycated proteins. Their formation and accumulation can lead to complications of diabetes, Alzheimer's disease, cataracts, arteriosclerosis, and other diseases, and are also a cause of aging and functional decline in tissues such as the skin. Therefore, the aforementioned oxidized proteins and glycated proteins are called damaging proteins. Among them, the accumulation of AGEs (Advanced Glycation End Products), the final products of the non-enzymatic addition reaction between sugars and proteins, is considered a problem.

[0003] AGEs (Advanced Glycation End Products) are brown in color. With age, their long-term accumulation in the skin leads to yellowing, dullness, and even the formation of spots, causing damage known as "skin glycation." Collagen and elastin are two key proteins found in connective tissue; they work together to maintain skin firmness and elasticity. AGEs break down collagen and elastin in the skin, leading to wrinkles and sagging. Furthermore, excessive sugar intake increases insulin release, further increasing androgen secretion, stimulating sebum production, clogging pores, and causing acne and pimples.

[0004] If we could clearly understand the degree of skin glycation, and assess the skin's condition based on that degree, we could more effectively choose suitable cosmetics or skincare products to prevent skin glycation, alleviate yellowing, dullness, and blemishes, and prevent skin aging, acne, and blackheads. Therefore, there is a greater need for more convenient and accurate methods for evaluating the degree of skin glycation, as well as devices for detecting it. Summary of the Invention

[0005] The technical problem that the invention aims to solve

[0006] In existing technologies, the determination of AGEs content is mostly done by measuring blood samples, because the purpose of measuring AGEs content in current technologies is related to disease treatment, and blood sample testing is more accurate. However, for the non-medical beauty and skincare industry that only needs to determine the degree of skin glycation and to care for the skin based on the degree of skin glycation, such as anti-glycation and anti-aging, it is very cumbersome to determine the level of AGEs in blood. Therefore, there is an urgent need for a simple and accurate method to evaluate the degree of skin glycation, as well as a device for detecting the degree of skin glycation.

[0007] Problem-solving methods

[0008] To address the aforementioned problems, the inventors conducted repeated and in-depth research on methods for evaluating the degree of skin glycation and devices for detecting the degree of skin glycation. As a result, they discovered that the following specific methods and devices can be used to evaluate the degree of skin glycation simply, quickly, and accurately, thus completing this invention.

[0009] That is, the present invention includes the following solutions:

[0010] 1. A method for evaluating the degree of skin glycation, characterized by comprising the following steps:

[0011] (1) Skin sample collection procedure: Take a sample from the skin by attaching it with tape;

[0012] (2) Skin sample extraction steps: Extract test samples from the sampling tape using Tris-HCl buffer and perform pretreatment;

[0013] (3) Skin sample identification procedure: Use LC-MS / MS external standard method, T3 column, to detect carboxymethyl lysine (CML) or pentosine;

[0014] (4) Determine the level of AGEs and the degree of glycation of the skin based on the detected levels of carboxymethyl lysine (CML) or pentosyl glycoside.

[0015] 2. The method for evaluating the degree of skin glycation as described in Scheme 1, characterized in that the detection time of the LC-MS / MS external standard method is 4 to 5 minutes.

[0016] 3. The method for evaluating the degree of skin glycation according to Scheme 1, characterized in that the detection limit of carboxymethyl lysine (CML) is 0.07 ng / mL and the detection limit of pentosyl glycoside is 0.03 ng / mL.

[0017] 4. A device for detecting the degree of skin glycation, characterized in that it comprises the following units:

[0018] (1) A sampling unit comprising a container for treating the adhesive tape sampled from the skin with Tris-HCl buffer, and a stage for holding the pretreated sample taken from the adhesive tape.

[0019] (2) LC-MS / MS unit: This unit uses the external standard method with a T3 column to detect carboxymethyl lysine (CML) or pentoglycosides to determine the AGEs content.

[0020] (3) Display unit, which visually displays the degree of saccharification based on the detected AGEs content.

[0021] 5. The skin glycation degree detection device according to Scheme 4, characterized in that the detection time of the LC-MS / MS unit using the external standard method is 4 to 5 minutes.

[0022] 6. The skin glycation degree detection device according to Scheme 4 is characterized in that the detection limit of carboxymethyl lysine (CML) is 0.07 ng / mL and the detection limit of pentosyl glycoside is 0.03 ng / mL.

[0023] Invention Effects

[0024] By using the skin glycation degree evaluation method and skin glycation degree detection device of the present invention, the skin glycation degree can be evaluated conveniently, quickly and accurately. Based on the skin glycation degree evaluation results obtained by the method or detection device of the present invention, suitable skin care products can be selected more specifically for skin care, resulting in better skin condition. Attached Figure Description

[0025] Figure 1 This is a graph showing the HPLC detection of CML standard (2.5 μg / mL).

[0026] Figure 2 These are the LC-MS / MS mass spectra of samples D7 and A2.

[0027] Figure 3 These are the LC-MS / MS mass spectra of samples D8 and A3. Detailed Implementation

[0028] The method for evaluating the degree of skin glycation and the device for detecting the degree of skin glycation involved in this invention will now be described in more detail.

[0029] The method for evaluating the degree of skin glycation involved in this invention is characterized by comprising the following steps:

[0030] (1) Skin sample collection procedure: Take a sample from the skin by attaching it with tape;

[0031] (2) Skin sample extraction procedure: Extract the test sample from the sampling tape using Tris-HCl buffer and perform pretreatment.

[0032] (3) Skin sample identification procedure: Use LC-MS / MS external standard method, T3 column, to detect carboxymethyl lysine (CML) or pentosine;

[0033] (4) Determine the level of AGEs and the degree of glycation of the skin based on the detected levels of carboxymethyl lysine (CML) or pentosyl glycoside.

[0034] Regarding step (1) in the skin glycation degree evaluation method of the present invention, it is done by using an adhesive tape method. Using adhesive tape (such as D100), non-invasive in vivo skin sampling was performed on subjects to attempt to establish an in vitro trace quantitative detection method for two AGEs components. Trace analysis refers to analytical methods where the content of the analyte in a sample is less than one part per million, i.e., μg / L. When using adhesive tape for skin sampling, there are no particular restrictions on the type of tape used; for example, D101 tape (model I-1QC-8CR3-1) or CuDerm brand D100 tape (model I-1QC-8CR2-0) can be used. A specific sampling method can be illustrated as follows: After cleaning the skin area to be sampled from the subject with water, wait 15-20 minutes; use D101 tape to remove dirt from the surface of the skin to be sampled; then take a piece of D100 tape, approximately 3.8 cm² in area. 2 It is adhered to the surface of the skin area to be sampled, and pressure is applied using a pressure rod at a pressure of 225 g / cm. 2 Press down for 10 seconds, repeating this pressing action 5 times to complete one piece of tape application. Repeat this step 4 times to obtain a total of 4 pieces of tape. Combine these 4 pieces of tape into one sample. If sampling is not performed according to this method, the concentration of the substance to be detected in the obtained skin sample may not meet the detection standard. Therefore, the sampling method in this invention is a simple and effective sampling method obtained by the applicant through repeated exploration and experimentation. If the number of samplings exceeds 4, it may adversely affect the skin barrier and cause skin damage. Therefore, the applicant also obtained a sampling method with minimal skin damage that achieves an effective sample concentration through repeated exploration and experimentation.

[0035] Regarding step (2) of the skin glycation degree evaluation method of the present invention, the test sample is extracted from the sampling tape using Tris-HCl buffer for pretreatment. The specific operation can be illustrated as follows: Roll up the adhesive side of the tape obtained in step (1) above, cut it into pieces with scissors, and put it into a 5mL EP tube; add 3mL of Tris-HCl buffer (50mmol / L, pH7.5) to the EP tube; centrifuge at 14000r / min for 15min, sonicate for 10min, and soak at room temperature for 4h; centrifuge again at 14000r / min for 15min, take the supernatant, blow with nitrogen and make up to 1mL as one test sample; store at -20℃, and the storage time should not exceed 2 weeks. If long-term storage is required, store at -80℃, and avoid repeated freeze-thaw cycles as much as possible, until testing.

[0036] Regarding step (3), the step of using LC-MS / MS external standard method to detect carboxymethyl lysine (CML) or pentoglycoside to evaluate the level of skin AGEs is a key step of the method of the present invention. Only by selecting the LC-MS / MS external standard method and the selected detection target carboxymethyl lysine (CML) or pentoglycoside can the purpose of the present invention be achieved.

[0037] To select suitable detection methods and appropriate analytes, the applicant conducted the following experiments: Enzyme-linked immunosorbent assay (ELISA) was used to detect carboxymethyl lysine (CML), pentosan, 3-deoxyglucuronide (3-DG), methylglyoxal (MGO), and carboxyethyl lysine (CEL); high-performance liquid chromatography (HPLC) was used to detect carboxymethyl lysine (CML); and liquid chromatography / mass spectrometry-mass spectrometry (LC / MS-MS) was used to detect carboxymethyl lysine (CML) and pentosan.

[0038] The applicant discovered that when using enzyme-linked immunosorbent assay (ELISA) for detection, the accuracy of the kit for testing exogenous samples was verified using blind samples (blind samples refer to a sample with a known true value used to test the accuracy of the analytical method; in this case, it refers to preparing a CML standard at a known concentration and using ELISA to check if it matches the known concentration). It was found that the tested concentration differed significantly from the given concentration, indicating poor accuracy, and the test values ​​at different concentration gradients did not show significant differences. The applicant speculates that the ELISA kit is suitable for detecting endogenous samples, such as urine and blood, and its accuracy is poor for exogenous samples. Therefore, this invention is not suitable for using enzyme-linked immunosorbent assay (ELISA).

[0039] When using HPLC to detect carboxymethyl lysine (CML) standards, it was found that the detection limit was high, making it unsuitable for testing on skin samples.

[0040] The concentration of the blind sample tested by liquid chromatography / mass spectrometry-mass spectrometry (LC / MS-MS method) is very close to that of the prepared blind sample. The method has good stability, high sensitivity, and low detection limit, and can detect two AGEs, carboxymethyl lysine (CML) and pentosolic acid, in skin samples.

[0041] Therefore, the method of this application, after skin sample extraction via adhesive tape, can detect carboxymethyl lysine (CML) and pentosine by LC-MS / MS. The accuracy results of the blind sample test method demonstrate that the quantitative results are highly accurate.

[0042] When using the LC-MS / MS external standard method, there are no particular restrictions on the choice of columns, but T3 columns are preferred.

[0043] In addition, there is no particular limitation on the detection time using the LC-MS / MS external standard method, but 4-5 minutes is preferred.

[0044] When using the LC-MS / MS external standard method to detect carboxymethyl lysine (CML) and pentoglycoside, the detection limit for CML was 0.07 ng / mL, and the detection limit for pentoglycoside was 0.03 ng / mL.

[0045] Regarding step (4), the degree of skin glycation is determined based on the skin AGEs level detected in step (3).

[0046] The inventors of this application have found the correlation between different AGEs components and the skin condition of subjects through experiments. By testing carboxymethyl lysine (CML) and pentosyl glycoside, the skin condition of subjects can be inferred to a certain extent.

[0047] The inventors discovered that the following physiological parameters showed a significant negative correlation (p<0.05) with carboxymethyl lysine (CML) content (measured by LC-MS / MS): stratum corneum moisture content (lower CML content corresponds to higher skin moisture content); and skin elasticity (lower CML content corresponds to higher skin elasticity). The following physiological parameters also showed a significant positive correlation (p<0.05) with CML content (measured by LC-MS / MS): skin yellowness (higher CML content corresponds to higher skin yellowness); and skin wrinkles (higher CML content corresponds to higher wrinkle severity). Furthermore, the following physiological parameter showed a significant positive correlation (p<0.05) with pentoglycoside content (measured by LC-MS / MS): transepidermal water loss (TEWL, a commonly used indicator of stratum corneum barrier function); higher pentoglycoside content corresponds to poorer skin barrier function.

[0048] Furthermore, the verification showed that carboxymethyl lysine (CML) and pentose glycosides detected by this method are significantly correlated with physiological parameters related to skin glycation, further demonstrating that CML and pentose glycosides detected by this method can serve as effective indicators for evaluating the degree of skin glycation.

[0049] Using the method of the present invention described above, skin samples can be easily taken to determine the level of AGEs and the degree of glycation. Based on the detected level of AGEs and the degree of glycation, appropriate cosmetics or skin care products can be selected to care for the skin in order to obtain a better skin condition.

[0050] The present invention provides a skin glycation degree detection device, characterized in that it comprises the following units:

[0051] (1) A sampling unit comprising a container for treating the adhesive tape sampled from the skin with Tris-HCl buffer, and a stage for holding the pretreated sample taken from the adhesive tape.

[0052] (2) LC-MS / MS unit: This unit uses the external standard method with a T3 column to detect CML or pentoglycosides to determine the AGEs content.

[0053] (3) Display unit, which visually displays the degree of saccharification based on the detected AGEs content.

[0054] The tape, reagents, etc. used in the above sampling unit can be the same as those described in the above method.

[0055] The LC-MS / MS unit described above can be the same as the LC-MS / MS unit used in the method described above.

[0056] There are no particular limitations on the display unit mentioned above; any display device that can display the detection results of this invention or visually display the degree of saccharification is acceptable.

[0057] With the skin glycation degree detection device of the present invention, the skin glycation degree can be easily determined by taking a skin sample. Based on the detected skin glycation degree, appropriate cosmetics or skin care products can be selected to care for the skin in order to obtain a better skin condition.

[0058] Example

[0059] The present invention is illustrated in detail below with examples, but the present invention is not limited to these examples. Those skilled in the art can make appropriate modifications based on the content of the examples without affecting the effects of the present invention, and such modifications are also within the scope of the present invention.

[0060] Example 1: Non-invasive in vivo sampling and quantitative detection of AGEs components

[0061] The determination shall be performed using the test reagents listed in Table 1 and the equipment listed in Table 2, in accordance with the following method.

[0062] Table 1 Test Reagents

[0063]

[0064]

[0065] Table 2 Test Equipment

[0066]

[0067]

[0068] Test methods

[0069] 1. Skin sample collection

[0070] (1) After washing their face with water, the subjects waited for 15-20 minutes.

[0071] (2) Use D101 tape to remove dirt from the subject's facial skin surface;

[0072] (3) Take a piece of D100 tape (3.8cm²) 2 It was applied to the skin of the subject's cheek and then subjected to pressure (225g / cm). 2 Press for 10 seconds, repeat the pressure bar pressing operation 5 times to complete the pasting of one piece of tape. Repeat this step 4 times to obtain 4 pieces of tape. Use the 4 pieces of tape as a sample.

[0073] (4) Roll up the adhesive side of the tape, cut it into pieces with scissors, and put it into a 5mL EP tube;

[0074] (5) Add 3 mL of Tris-HCl buffer (50 mmol / L, pH 7.5) to the EP tube;

[0075] (6) Centrifuge at 14000 r / min for 15 min, sonicate for 10 min, and soak at room temperature for 4 hours;

[0076] (7) Centrifuge again at 14000r / min for 15min, take the supernatant, blow it with nitrogen and make up to 1mL as a test sample;

[0077] (8) Store at -20℃ (do not store for more than 2 weeks; if long-term storage is required, store at -80℃ and avoid repeated freeze-thaw cycles as much as possible) and wait for testing.

[0078] Example 2. Enzyme-linked immunosorbent assay (ELISA) detection of carboxymethyl lysine (CML), pentosine, 3-deoxyglucuronide (3-DG), methylglyoxal (MGO), and carboxyethyl lysine (CEL);

[0079] Enzyme-linked immunosorbent assay (ELISA) is a novel immunoassay technique developed based on enzyme immunoassay technology.

[0080] Currently, there are many ELISA kits on the market for detecting glycation-related indicators. Research shows that kits are available for carboxymethyl lysine (CML), pentosyl glycoside, 3-deoxyglucuronide (3-DG), methylglyoxal (MGO), and carboxyethyl lysine (CEL). However, these kits generally have low limits of detection and quantification, and are suitable for endogenous samples such as serum, urine, and plasma. No kits designed for exogenous samples were found, and the accuracy of their detection for exogenous samples is unknown, with no published literature supporting this accuracy. In this experiment, to test the accuracy of the kit in quantitatively detecting exogenous samples, carboxymethyl lysine (CML) was selected as the assay. Blind samples of CML standards at different concentrations were prepared and tested to determine the accuracy of the ELISA method in determining the CML content in exogenous samples (skin samples).

[0081] Feasibility verification results of ELISA method

[0082] Table 3. Results of ELISA kit testing non-self CML standards

[0083]

[0084] The above results indicate that ELISA is not accurate for detecting foreign samples, and the matrix effect significantly impacts the results. For example, the concentration of the blind standard was 4 ng / mL, while the concentration measured by the ELISA kit was 16.35 ng / mL, resulting in a large deviation in the data measured by the ELISA method. Furthermore, the matrix from skin samples has an even more complex composition, which may significantly affect the accuracy of the experimental results.

[0085] Example 3 uses high performance liquid chromatography (HPLC) to detect carboxymethyl lysine (CML);

[0086] High-performance liquid chromatography (HPLC), also known as high-pressure liquid chromatography, high-speed liquid chromatography, high-resolution liquid chromatography, etc., is also called chromatography or chromatography. HPLC has a wide range of applications and is suitable for a broad range of samples. It is not limited by the volatility and thermal stability of the analyte. Almost all compounds, including high-boiling-point, polar, ionic compounds and macromolecules, can be analyzed and determined by HPLC. Its instrument detection limit can generally reach ng / ml, making it suitable for establishing trace detection methods. In this experiment, carboxymethyl lysine (CML) was chosen (based on previous literature review, it has the highest probability of detection) for method establishment.

[0087] Carboxymethyl lysine (CML) does not exhibit fluorescence properties, therefore it is detected using a UV detector. To test the limit of quantitation and limit of detection of CML samples by HPLC, a certain concentration of CML standard was prepared for testing.

[0088] Feasibility verification results of HPLC method for detecting carboxymethyl lysine (CML)

[0089] The HPLC method for detecting carboxymethyl lysine (CML) standard showed that at a concentration of 2.5 μg / ml, it eluted at 10 min, exhibiting an inverted peak shape. The chromatogram is shown below. Figure 1 As shown, it was not detected, therefore the HPLC method for quantitative detection of carboxymethyl lysine (CML) was abandoned.

[0090] Example 4 uses liquid chromatography / mass spectrometry-mass spectrometry (LC / MS-MS) to detect carboxymethyl lysine (CML);

[0091] LC-MS / MS is widely used in various fields such as food safety, environmental monitoring, and impurity analysis. The basic principle is the coupling of liquid chromatography and secondary mass spectrometry. Through liquid phase separation and mass spectrometry ionization, the analytical purpose is achieved by measuring the intensity of various ion spectral peaks. It can perform qualitative and quantitative detection, and its sensitivity and detection limit are both high.

[0092] To test the detection limit, quantitation limit, and accuracy of LC-MS / MS for determining samples, blind samples of CML standard at a certain concentration and skin samples of subjects were prepared for testing to determine the feasibility of establishing a trace quantitative detection method using LC-MS / MS for these test samples.

[0093] Test sample A

[0094] (1) Blind sample of carboxymethyl lysine CML standard, labeled D7: 2.5 ng / mL,

[0095] (2) Subject number: A2

[0096] B Test Method

[0097] 1. Preparation of standard solutions

[0098] Standard solutions were prepared by serial dilution: 0.50 ng / mL, 1.00 ng / mL, 2.00 ng / mL, 5.00 ng / mL, 10.00 ng / mL, 20.00 ng / mL, 50.00 ng / mL, 80 ng / mL, 100.00 ng / mL, and 200.00 ng / mL.

[0099] 2. Instruments and equipment

[0100] Chromatographic column: HSS T3 1.8μm (2.1×100mm)

[0101] Equipment Model: AB SCIEX TRipley Quad™ 5500+System

[0102] 3. Testing conditions

[0103] (1) Test conditions

[0104] As shown in Table 4

[0105] Table 4. Test conditions for the determination of carboxymethyl lysine (CML) by LC-MS / MS.

[0106]

[0107] (2) Chromatographic conditions:

[0108] Mobile phase: water and acetonitrile (containing 0.1% formic acid), flow rate: 0.5 mL / min, injection volume: 10 μL; gradient ratio: water:acetonitrile:initial ratio: 85:15, 0.5 min: 85:15, 2.0 min: 5:95, 3.5 min: 5:95, 3.6 min: 90:10, 4.6 min: 90:10.

[0109] (3) Mass spectrometry conditions:

[0110] Curtain Gas (CUR): 40, IonSpray Voltage (IS): 5500, Temperature (TEM): 550℃, Collision Gas (CAD): 7, Ion Source Gas1:50, Ion Source Gas2:50

[0111] (4) Feasibility verification results of LC-MS / MS method for determining carboxymethyl lysine (CML)

[0112] Linear relationship: y = 7.45 × 10 4 x + 1.66 × 10 4 (R 2 =0.9991, R² ranges from 0 to 1. The larger the R² value, the better the model fit, i.e., the greater the explanatory power of the model. If the R² value is equal to 1, it means that the model perfectly fits the data; if the R² value is close to 0, it means that the model has almost no explanatory power for the data variability. The detection limit is 0.07 ng / mL, the quantitation limit is 0.21 ng / mL, the recovery rate is between 99% and 109%, the intra-sample precision RSD is less than 5.46%, the inter-sample precision RSD is less than 4.55%, and the method has good stability.

[0113] The content of the blinded standard sample was highly correlated with the content of the prepared standard, indicating high accuracy. The results of the blinded sample D7 test and the results of the subject's skin sample A2 test are shown in Table 5; the mass spectra are shown below. Figure 2As shown, carboxymethyl lysine (CML) can be detected by LC-MS / MS, indicating that the skin samples collected and extracted using the tape adhesion method established in this study can be detected by in vitro detection methods.

[0114] Table 5 Results of quantitative determination of carboxymethyl lysine (CML) by LC-MS / MS method

[0115]

[0116] Example 5: Feasibility Verification of Quantitative Detection of Pentoglobin by LC-MS / MS Method

[0117] To test the detection limit, quantitation limit, and accuracy of LC-MS / MS in determining samples, a blind sample of pentosolic acid standard at a certain concentration was prepared and a skin sample was extracted from one subject for testing. The feasibility of establishing a trace quantitative detection method using LC-MS / MS for this test sample was determined.

[0118] Test sample A

[0119] (1) Pentoside standard blind sample, labeled D8: 1 ng / mL,

[0120] (2) Subject number: A3

[0121] B Test Method

[0122] 1. Preparation of standard solutions

[0123] Standard solutions were prepared using a stepwise dilution method: 0.10 ng / mL, 0.20 ng / mL, 0.50 ng / mL, 1.00 ng / mL, 2.00 ng / mL, 5.00 ng / mL, 10.00 ng / mL, 20.00 ng / mL, 50.00 ng / mL, 100.00 ng / mL, and 200.00 ng / mL.

[0124] 2. Instruments and equipment

[0125] Chromatographic column: HSS T3 1.8μm (2.1×100mm)

[0126] Equipment Model: AB SCIEX TRipley Quad TM 5500+System

[0127] 3. Testing conditions

[0128] (1) Chromatographic conditions

[0129] Mobile phase: water and acetonitrile (containing 0.1% formic acid), flow rate: 0.5 mL / min, injection volume: 10 μL, gradient ratio: water:acetonitrile:initial ratio: 90:10, 0.5 min: 90:10, 2.5 min: 5:95, 3.5 min: 5:95, 3.6 min: 90:10, 4.6 min: 90:10.

[0130] (2) Mass spectrometry conditions

[0131] Curtain Gas (CUR): 40, IonSpray Voltage (IS): 5500, Temperature (TEM): 550℃, Collision Gas (CAD): 7, Ion Source Gas1:50, Ion Source Gas2:50DP:91V, EP:10V, CE:25V.

[0132] 4. Detection results of pentoglycosides

[0133] Linear relationship: y = 1.0971x - 0.9154 (R) 2 =0.9997), detection limit: 0.03 ng / mL, quantitation limit: 0.1 ng / mL, recovery rate between 80% and 109%, intra-sample precision RSD less than 8.31%, inter-sample precision RSD less than 11.12%, this method has good stability.

[0134] The content of the blinded standard sample was highly correlated with the content of the prepared standard sample, indicating high accuracy. The results of the blinded sample D8 test and the results of the subject's skin sample A3 test are shown in Table 6; the mass spectra are shown below. Figure 3 As shown, pentoglycosides can be detected by LC-MS / MS, indicating that the skin samples collected and extracted using the tape adhesion method established in this study can be detected by in vitro detection methods.

[0135] Table 6. Results of LC-MS / MS testing of pentoglycoside standard in blind samples.

[0136]

[0137] The experimental results from the above embodiments lead to the following conclusions:

[0138] (1) ELISA method: The accuracy of foreign samples was tested using a blind sample verification kit. It was found that the test concentration differed greatly from the given concentration, resulting in poor accuracy. Moreover, the test values ​​under different concentration gradients did not differ significantly. The reason is speculated to be that the ELISA kit is suitable for the detection of endogenous samples, such as urine and blood, and has poor accuracy for foreign samples.

[0139] (2) HPLC method: The standard for detecting carboxymethyl lysine (CML) has a high detection limit and is not suitable for testing on skin samples. Therefore, it is recommended to abandon this method.

[0140] (3) LC-MS / MS method: The concentration of the blind sample tested is close to that of the prepared blind sample. The method has good stability, high sensitivity, and low detection limit. It can detect two AGEs, carboxymethyl lysine (CML) and pentosine, in skin samples.

[0141] (4) The tape application method established in this study can detect CML and pentosolic acid by LC-MS / MS after skin sample extraction. The results of the blind sample test show that the quantitative results are highly accurate.

[0142] As can be seen from the detection results of the above embodiments, although ELISA and HPLC are also commonly used detection methods, these two methods cannot accurately detect the content levels of carboxymethyl lysine (CML) and pentoglycosides in the samples of this invention. Only the LC-MS / MS method used in this invention can accurately detect the levels of carboxymethyl lysine (CML) and pentoglycosides.

[0143] Example 6. Correlation analysis between different AGEs components and subject conditions

[0144] 1. Experimental Objective

[0145] Establish the correlation between different AGEs components (types and contents) and subject conditions (skin physiological parameters).

[0146] 2. Subjects, equipment and instruments

[0147] (1) Subjects: 30 female volunteers aged 20-59 were recruited.

[0148] (2) Test instruments: as shown in Table 7.

[0149] Table 7 Test Instruments

[0150]

[0151] 3. Test Methods

[0152] (1) Collect skin samples from volunteers and detect AGEs components: CML and pentosolic acid were detected by ELISA and LC-MS / MS respectively.

[0153] (2) Collect volunteer skin physiological parameters: stratum corneum moisture content, transepidermal water loss (TEWL), Lab value, ITA° value, skin wrinkles, elasticity parameters, and skin VISIA; TEWL represents the physical parameter of normal and continuous diffusion of water through the stratum corneum, used to evaluate skin barrier function. When the skin barrier is damaged, the TEWL value tends to increase. Lab color space, tristimulus colorimeter is used for skin color measurement, thus evolving the investigation of human skin color from subjective evaluation to objective quantification. L* value reflects skin brightness, a* value reflects skin redness, the higher the a* value, the redder the skin; b* value reflects skin yellowness, the higher the b* value, the yellower the skin. ITA° is calculated by the formula ITA°=[Arc Tan(L*-50) / b*]×180 / 3.14159, which can reflect the whiteness of the skin.

[0154] 4. Analysis of different AGEs components and volunteer characteristics: SPSS (Statistical Program for Social Sciences) software was used to analyze the correlation between skin physiological parameters and AGEs components. The significance level was set at α = 0.05.

[0155] Results Analysis and Discussion

[0156] 4.1 Correlation analysis between skin physiological parameters and AGEs components

[0157] (1) No correlation was found between the content of CML (ELISA method) and pentosyl glycoside (ELISA method) and skin physiological parameters;

[0158] (2) Physiological parameters that showed a significant negative correlation (p<0.05) with carboxymethyl lysine (CML) content (determined by LC-MS / MS) included: stratum corneum moisture content, i.e., the lower the CML content, the higher the stratum corneum moisture content; and skin elasticity, i.e., the lower the CML content, the higher the skin elasticity. Physiological parameters that showed a significant correlation (p<0.05) with CML content (determined by LC-MS / MS) included: skin yellowness, i.e., the higher the CML content, the higher the skin yellowness; and skin wrinkles, i.e., the higher the CML content, the higher the degree of skin wrinkles.

[0159] (3) No physiological parameters were found to be significantly negatively correlated with pentoglycoside content (as determined by LC-MS / MS); the physiological parameters that were found to be significantly positively correlated with pentoglycoside content (as determined by LC-MS / MS) (p<0.05) were: TEWL, that is, the higher the pentoglycoside content, the worse the skin barrier function.

[0160] 5. Summary

[0161] (1) There is a significant correlation between skin physiological parameters and AGEs components. The content of carboxymethyl lysine (CML) is significantly correlated with the moisture content of the stratum corneum, skin elasticity, skin yellowness and skin wrinkle degree; the content of pentosyl glycoside is significantly correlated with TEWL. Therefore, it is recommended to study the correlation between skin physiological parameters and AGEs components.

[0162] (2) Although there is no research on applying LC-MS / MS to the evaluation of multiple skin characteristics, the above research results show that this method can provide a skin evaluation method that can evaluate multiple different skin test indicators using a single test, as well as a method for recommending skin care products or cosmetics.

[0163] Industrial availability

[0164] By using the skin glycation degree evaluation method and skin glycation degree detection device of the present invention, it is possible to simply take skin samples to determine the degree of skin glycation. Based on the detected degree of skin glycation, appropriate cosmetics or skin care products can be selected to care for the skin in order to obtain a better skin condition.

Claims

1. A method for evaluating the degree of skin glycation, characterized in that, Includes the following steps: (1) Skin sample collection procedure: Take a sample from the skin by attaching it with tape; (2) Skin sample extraction steps: Extract test samples from the sampling tape using Tris-HCl buffer and perform pretreatment; (3) Skin sample identification procedure: LC-MS / MS external standard method, using a T3 column, to detect CML or pentoglobin; (4) Determine the level of AGEs and the degree of glycation of the skin based on the detected carboxymethyl lysine (CML) or pentosyl glycoside content.

2. The method for evaluating the degree of skin glycation according to claim 1, characterized in that, The detection time for the LC-MS / MS external standard method is 4-5 minutes.

3. The method for evaluating the degree of skin glycation according to claim 1, characterized in that, The detection limit for carboxymethyl lysine (CML) was 0.07 ng / mL, and the detection limit for pentosolic acid was 0.03 ng / mL.

4. A device for detecting the degree of skin glycation, characterized in that, Includes the following units: (1) A sampling unit comprising a container for treating the adhesive tape sampled from the skin with Tris-HCl buffer, and a stage for holding the pretreated sample taken from the adhesive tape. (2) LC-MS / MS unit: This unit uses the external standard method with a T3 column to detect CML or pentoglycosides to determine the AGEs content. (3) Display unit, which visually displays the degree of saccharification based on the detected carboxymethyl lysine (CML) or pentoside content.

5. The skin glycation degree detection device according to claim 4, characterized in that, The detection time for the LC-MS / MS unit using the external standard method is 4-5 minutes.

6. The skin glycation degree detection device according to claim 4, characterized in that, The detection limit for CML was 0.07 ng / mL, and the detection limit for pentosolic acid was 0.03 ng / mL.

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