Detection method of blue copper peptide related substances

Abstract

The invention belongs to the field of cosmetics, and particularly relates to a blue-copper peptide related substance detection method which adopts a high performance liquid chromatograph for detection, and the chromatographic conditions are as follows: a chromatographic column is a reversed-phase C18 or C8 reversed-phase chromatographic column; a mobile phase: phosphate; the wavelength of an ultraviolet detector is 220nm; the column temperature is 25-40 DEG C; the flow velocity of the mobile phase is 0.5-1.0 ml / min; the elution procedure is equal-gradient elution. The chromatographic column in the detection method of the blue-copper peptide related substance can realize separation of the ligand compound and the blue-copper peptide, the separation reaches 1.5, and qualitative and quantitative accurate detection of the blue-copper peptide related substance can be realized based on the excellent separation effect of the chromatographic column.

Description

Technical Field

[0001] The invention belongs to the field of cosmetics, and particularly relates to a method for detecting blue copper peptide-related substances. Background Art

[0002] Blue copper peptide, also known as blue copper peptide, copper peptide, tripeptide-1 copper, abbreviated as GHK-Cu in English, has a rare structural sequence of glycyl-L-histidyl-L-lysine-Cu (Ⅱ). Tripeptide-1 is a small molecule protein composed of three amino acids. Copper is a trace element required to maintain body functions, and blue copper peptide is a complex of metal copper ions and tripeptides. Blue copper peptide has the dual functions of signal peptide and carrier peptide, and is called the "Hermes" among small molecule peptides. Its main functions are to promote cell regeneration, improve skin pigmentation, enhance skin immunity, and resist oxidation. Blue copper peptide achieves the purpose of repairing skin and delaying aging by strengthening the effect of copper ions on oxides. However, too much copper ions can damage cells and accelerate skin aging. Therefore, the chelation of copper ions and tripeptide-1 is the most critical factor for whether blue copper peptide can function. Tripeptide-1 that is not chelated to form blue copper peptide becomes an impurity in the raw material of blue copper peptide, that is, a ligand compound.

[0003] The commonly used detection method for blue copper peptide and its impurities (ie, detection of complexes) is: using reverse phase chromatography with a 0.1% trifluoroacetic acid system as the mobile phase, but there are cases where the blue copper peptide is weakly retained and the ligand compound is not well separated from the main component blue copper peptide.

[0004] The existing technology for liquid phase detection of blue copper peptides discloses a liquid phase detection method for the content of blue copper peptides. Although the content of blue copper peptides in cosmetics can be accurately measured, tripeptide-1 in the raw material of blue copper peptides and its purity cannot be measured. Summary of the invention

[0005] In order to solve the problem that tripeptide-1 and its purity cannot be detected in the prior art, the present invention provides a method for detecting blue copper peptide-related substances.

[0006] The detection method of the blue copper peptide related substances adopts high performance liquid chromatography for detection, and the chromatographic conditions are as follows:

[0007] Chromatographic column: reverse phase C18 or C8 reverse phase column;

[0008] Mobile phase: phosphate;

[0009] UV detector wavelength: 220nm;

[0010] Column temperature: 25-40°C;

[0011] Mobile phase flow rate: 0.5~1.0mL / min;

[0012] Elution program: isocratic elution.

[0013] Furthermore, the phosphate is selected from one or more of ammonium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

[0014] Furthermore, the phosphate is potassium dihydrogen phosphate.

[0015] Furthermore, the phosphate concentration may be 10 mM to 40 mM.

[0016] Furthermore, the phosphate concentration is 10 mM.

[0017] Furthermore, the pH value of the phosphate may be 7.0 to 9.0.

[0018] Furthermore, the pH value of the phosphate is 8.5.

[0019] Furthermore, the flow rate of the mobile phase is 1.0 mL / m, the column temperature is 35° C., and the chromatographic column is a C18 chromatographic column.

[0020] Furthermore, the filling particle size of the chromatographic column is in the micron level, and the chromatographic column is a chromatographic column resistant to pure water phase.

[0021] Furthermore, the filling particle size of the chromatographic column is 5 microns, and the chromatographic column is a T3 chromatographic column.

[0022] Beneficial effects:

[0023] The chromatographic column in the method for detecting blue copper peptide related substances provided by the present invention can separate the ligand compound from the blue copper peptide, and the separation ratio reaches 1.5. Based on the excellent separation effect of the chromatographic column, accurate qualitative and quantitative detection of blue copper peptide related substances can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the UV chromatogram of Example 1 in which the method was applied to detect the impurity tripeptide-1 containing the ligand compound.

[0025] Figure 2 This is the mass spectrometry detection result of impurities enriched by this method in Example 1.

[0026] Figure 3 This is the UV chromatogram of Example 2 in which the method was applied to detect the impurity tripeptide-1 containing the ligand compound.

[0027] Figure 4 This is the UV chromatogram of Example 3 in which the method was used to detect the impurity tripeptide-1 containing the ligand compound.

[0028] Figure 5 The results of detecting related substances of blue copper peptide in Comparative Example 1 are shown.

[0029] Figure 6 This is the mass spectrometry detection result of the enriched impurities in Comparative Example 1. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below with reference to the accompanying drawings, but they should not be construed as limiting the applicable scope of the present invention.

[0031] According to the provisions of 0512 High Performance Chromatography in the Pharmacopoeia of the People's Republic of China:

[0032] The separation R is used to evaluate the separation degree between the test substance and the separated substance, and is a key indicator for measuring the separation efficiency of the chromatographic system. The separation efficiency of the chromatographic system can be evaluated and adjusted by measuring the separation between the test substance and known impurities, or by measuring the separation between the test substance and a certain index component (internal standard substance or other difficult-to-separate substance), or by degrading the test substance or reference substance by appropriate methods and measuring the separation between the test substance and a certain degradation product.

[0033] Whether it is a qualitative determination or a quantitative determination, it is required that there is a good separation between the chromatographic peak of the substance to be measured and the chromatographic peak of the internal standard substance or the chromatographic peak of a specific impurity control and other chromatographic peaks. Unless otherwise specified, the separation between the chromatographic peak of the substance to be measured and the adjacent chromatographic peak should not be less than 1.5. The calculation formula for the separation is:

[0034]

[0035] Where, t R2 is the retention time of the latter of two adjacent chromatographic peaks;

[0036] t R1 is the retention time of the previous peak between two adjacent chromatographic peaks;

[0037] W1, W2 and W 1,h / 2 , W 2,h / 2 are the peak width and half-peak width of the adjacent chromatographic peak respectively.

[0038] The present invention uses the separation degree R to evaluate the separation degree between the blue copper peptide and the tripeptide-1, so as to measure the separation efficiency of the chromatographic system involved in the detection method of the blue copper peptide related substance provided by the present invention. If the separation efficiency reaches the above provisions, the detection method of the blue copper peptide related substance provided by the present invention has a high accuracy. The related substance can be the main component blue copper peptide or the impurity tripeptide-1.

[0039] The present invention provides a method for detecting blue copper peptide related substances, wherein the method for detecting blue copper peptide related substances adopts a high performance liquid chromatograph for detection, and the chromatographic conditions are as follows:

[0040] Chromatographic column: reverse phase C18 or C8 reverse phase column;

[0041] Mobile phase: phosphate;

[0042] UV detector wavelength: 220nm;

[0043] Column temperature: 25-40°C;

[0044] Mobile phase flow rate: 0.5~1.0mL / min;

[0045] Elution program: isocratic elution.

[0046] Optionally, the phosphate is selected from one or more of ammonium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

[0047] Preferably, the phosphate is potassium dihydrogen phosphate.

[0048] Optionally, the phosphate concentration may be 10 mM to 40 mM.

[0049] Preferably, the phosphate concentration is 40 mM.

[0050] Optionally, the pH value of the phosphate may be 7.0-9.0.

[0051] Preferably, the pH value of the phosphate is 8.5.

[0052] Preferably, the mobile phase flow rate is 1.0 mL / m, the column temperature is 35° C., and the chromatographic column is a C18 chromatographic column.

[0053] Furthermore, the filling particle size of the chromatographic column is in the micron level, and the chromatographic column is a chromatographic column resistant to pure water phase.

[0054] Preferably, the filling particle size of the chromatographic column is 5 microns, and the chromatographic column is a T3 chromatographic column.

[0055] Example 1

[0056] Pretreatment of the sample to be tested: Take an appropriate amount of the sample, dissolve it in water, and dilute it to make a solution containing about 1 mg of blue copper peptide per 1 mg. Test conditions:

[0057] (a) Instrument: Agilent high performance liquid chromatograph;

[0058] (b) Chromatographic column: Waters HSS T3 4.6 mm × 250 mm, 5 μm;

[0059] (c) Mobile phase: isocratic elution; 40 mM potassium dihydrogen phosphate (adjusted to pH 8.5 with 10 mM potassium hydroxide);

[0060] (d) Column temperature: 35 °C;

[0061] (e) Flow rate: 1.0 mL / min;

[0062] (f) Injection volume: 10 μl;

[0063] (g) Running time: 30 minutes;

[0064] (h) UV detector wavelength: 220 nm.

[0065] The separated related substances were detected by mass spectrometry.

[0066] Embodiment 2:

[0067] Pretreatment of the sample to be tested: Take an appropriate amount of the sample, dissolve it in water, and dilute it to make a solution containing about 1 mg of blue copper peptide per 1 mg. Test conditions:

[0068] a) Instrument: Agilent high performance liquid chromatograph;

[0069] b) Chromatographic column: Waters HSS T3 4.6 mm × 250 mm, 5 μm;

[0070] c) Mobile phase: isocratic elution; 20 mM potassium dihydrogen phosphate (adjust pH to 8.5 with 10 mM potassium hydroxide);

[0071] d) Column temperature: 35°C;

[0072] e) Flow rate: 1.0 mL / min;

[0073] f) Injection volume: 10 μl;

[0074] g) Running time: 30 minutes;

[0075] h) UV detector wavelength: 220nm.

[0076] The separated related substances were detected by mass spectrometry.

[0077] Embodiment 3:

[0078] Pretreatment of the sample to be tested: Take an appropriate amount of the sample, dissolve it in water, and dilute it to make a solution containing about 1 mg of blue copper peptide per 1 mg. Test conditions:

[0079] a) Instrument: Agilent high performance liquid chromatograph;

[0080] b) Chromatographic column: Waters HSS T3 4.6 mm × 250 mm, 5 μm;

[0081] c) Mobile phase: isocratic elution; 40 mM potassium dihydrogen phosphate (adjust pH to 7.0 with 10 mM potassium hydroxide);

[0082] d) Column temperature: 35°C;

[0083] e) Flow rate: 1.0 mL / min;

[0084] f) Injection volume: 10 μL;

[0085] g) Running time: 30 minutes;

[0086] h) UV detector wavelength: 220nm.

[0087] Comparative Example 1

[0088] Pretreatment of the sample to be tested: Take an appropriate amount of the test sample, add the mobile phase to dissolve it, and dilute it to make a solution containing about 1 mg of blue copper peptide per 1 mg.

[0089] Testing conditions:

[0090] (a) Instrument: High performance liquid chromatography;

[0091] (b) Column: Waters XBridge BEH Amide, 4.6 mm × 75 mm, 2.5 μm;

[0092] (c) Mobile phase: isocratic elution; acetonitrile (0.1% trifluoroacetic acid) + water (0.1% trifluoroacetic acid) = 65 + 35; flow rate:

[0093] 0.4mL / min;

[0094] (d) Column temperature: 40 °C;

[0095] (e) Injection volume: 5 μL;

[0096] (f) Running time: 15 min;

[0097] (g) UV detector wavelength: 220 nm.

[0098] The separated related substances were detected by mass spectrometry.

[0099] Result analysis:

[0100] Figure 1 This is the analysis spectrum of blue copper peptide-related substances detected by the method in Example 1.

[0101] Table 1 shows the liquid phase detection results of blue copper peptides in Examples 1 to 3.

[0102] Table 1

[0103]

[0104] Figure 2 This is the mass spectrometry detection result of enriching the RT5.191min peak using this method in Example 1.

[0105] Figure 3 This is the analysis spectrum of related substances of blue copper peptide detected by the present method in Example 2.

[0106] Figure 4 This is the analysis spectrum of related substances of blue copper peptide detected by the present method in Example 3.

[0107] Result analysis: Figure 1-4 As shown in Table 1, the separation degree of blue copper peptide and tripeptide-1 can be greater than 1.5 in Examples 1 to 3, and complete separation can be achieved. Overall, the separation degree of the method of Implementation Case 1 is better. In Example 1, the peak with RT of 5.191min was enriched, and its molecular weight was 341.192Da through mass spectrometry detection, which is tripeptide-1. Therefore, the method can achieve the separation of blue copper peptide and its related substance tripeptide-1.

[0108] Figure 5 This is the analysis spectrum of related substances of blue copper peptide detected in Comparative Example 1.

[0109] Figure 6 This is the mass spectrometry detection result of the peak enriched at RT6.180min in Comparative Example 1.

[0110] Table 2 is the detection result of blue ketone peptide in comparative example 1.

[0111] Table 2

[0112] Serial number name Retention time min Peak area% Separation 1 Blue peptide 5.324 99.95％ / 2 / 6.180 0.05％ 0.8

[0113] In Comparative Example 1, the peak with an enriched RT of 6.180 min was detected by mass spectrometry, and its molecular weight was 402.1073 Da, which was a blue copper peptide. This peak was the tail of the blue copper peptide under the chromatographic conditions, and was not tripeptide-1.

[0114] Result analysis: Figure 5-6 As shown in the results of Table 2, the related substances of blue copper peptide (tripeptide-1) could not be separated in Comparative Example 1, indicating that Example 1 can realize the analysis of related substances of blue copper peptide.

Claims

1. A method for detecting blue copper peptide-related substances, characterized in that: High performance liquid chromatography was used for detection, and the chromatographic conditions were as follows: Chromatographic column: reverse phase C18 or C8 reverse phase column; Mobile phase: phosphate; UV detector wavelength: 220nm; Column temperature: 25-40°C; Mobile phase flow rate: 0.5~1.0mL / min; Elution program: isocratic elution.

2. The method for detecting blue copper peptide-related substances according to claim 1, characterized in that: The phosphate is selected from one or more of ammonium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

3. The method for detecting blue copper peptide-related substances as claimed in claim 2, characterized in that: The phosphate is potassium dihydrogen phosphate.

4. The method for detecting blue copper peptide-related substances according to claim 1, characterized in that: The phosphate concentration may be 10 mM to 40 mM.

5. The method for detecting blue copper peptide-related substances according to claim 4, characterized in that: The phosphate concentration was 10 mM.

6. The method for detecting blue copper peptide-related substances according to claim 1, characterized in that: The pH value of the phosphate may be 7.0 to 9.

0.

7. The method for detecting blue copper peptide-related substances according to claim 6, characterized in that: The pH value of the phosphate is 8.

5.

8. The method for detecting blue copper peptide-related substances according to claim 1, characterized in that: The flow rate of the mobile phase is 1.0 mL / m, the column temperature is 35° C., and the chromatographic column is a C18 chromatographic column.

9. The method for detecting blue copper peptide-related substances according to claim 1, characterized in that: The filling particle size of the chromatographic column is in the micron level, and the chromatographic column is a chromatographic column resistant to pure water phase.

10. The method for detecting blue copper peptide-related substances according to claim 9, characterized in that: The filling particle size of the chromatographic column is 5 microns, and the chromatographic column is a T3 chromatographic column.

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