A detection method for glycine palmitic acid dipeptide

Abstract

The present invention provides a method for detecting glycine palmitoyl dipeptide. The sample containing glycine palmitoyl dipeptide is detected by liquid chromatography, and the mobile phase includes a formic acid methanol solution and a formic acid aqueous solution. The detection method provided by the present invention can achieve accurate detection of glycine palmitoyl dipeptide. In addition, a methodological investigation is carried out on the detection method provided by the present invention. The experimental results show that the detection method has good system suitability, good precision, strong specificity, and high sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of chromatographic analysis, and particularly relates to a detection method for glycine palmitoyl dipeptide. Background Art

[0002] Palmitoyl tripeptide-1 is a messenger peptide for collagen renewal. It acts on the dermis layer, can stimulate the synthesis of collagen and glycosaminoglycans, strengthen the dermis layer, make the skin become more firm, soothe wrinkles, and has the effects of enhancing skin firmness and improving skin color. Therefore, palmitoyl tripeptide-1 is often used in the preparation of creams, lotions and essence products for anti-wrinkle, anti-aging, improving skin elasticity and moisture. At present, the preparation of palmitoyl tripeptide-1 is mainly through artificial synthesis.

[0003] Glycine palmitoyl dipeptide is an important dipeptide fragment for the segmented synthesis of palmitoyl tripeptide-1. The purity of glycine palmitoyl dipeptide directly determines the quality of palmitoyl tripeptide-1 products. Therefore, strict quality detection and synthesis control of glycine palmitoyl dipeptide are required. However, the prior art has not disclosed a detection method for glycine palmitoyl dipeptide. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a detection method for glycine palmitoyl dipeptide, which can accurately detect glycine palmitoyl dipeptide and is helpful for monitoring the quality of glycine palmitoyl dipeptide.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] A detection method for glycine palmitoyl dipeptide, using liquid chromatography to detect a sample containing glycine palmitoyl dipeptide, and the mobile phase includes a formic acid methanol solution and a formic acid aqueous solution.

[0007] Preferably, gradient elution is adopted during the detection process.

[0008] Preferably, the gradient elution program is as follows:

[0009]

[0010] Preferably, the volume concentration of formic acid in the formic acid methanol solution is 0.1-0.3%, and the volume concentration of formic acid in the formic acid aqueous solution is 0.1-0.3%.

[0011] Preferably, the flow rate of the mobile phase is 1.0-1.4 mL / min.

[0012] Preferably, the detection process further includes dissolving the sample containing glycine palmitoyl dipeptide with methanol.

[0013] Preferably, the above dissolution is carried out at a temperature of 30-70°C.

[0014] Preferably, in the detection process, the stationary phase of the chromatographic column used is C18, C16 or C30.

[0015] Preferably, the column temperature of the above-mentioned chromatographic column is 25-35 °C.

[0016] Preferably, in the detection process, an electrospray detector or an evaporative light scattering detector is used for detection.

[0017] Preferably, in the detection process, the injector temperature is 35-45 °C and the injection volume is 8-12 μL.

[0018] The present invention provides a detection method for glycine palmitate dipeptide. Using a formic acid methanol solution as the organic phase and a formic acid aqueous solution as the aqueous phase, liquid chromatography is used to detect a sample containing glycine palmitate dipeptide, which can achieve accurate detection of glycine palmitate dipeptide. The experimental results show that the detection method for glycine palmitate dipeptide provided by the present invention has good system suitability, good precision, strong specificity and high sensitivity, which is helpful for monitoring the quality of glycine palmitate dipeptide. Description of the Drawings

[0019] Figure 1 It is a chromatogram for detecting a sample containing glycine palmitate dipeptide with a formic acid methanol solution and a formic acid aqueous solution as the mobile phase;

[0020] Figure 2 It is a chromatogram for detecting an intermediate of glycine palmitate dipeptide with a formic acid methanol solution and a formic acid aqueous solution as the mobile phase;

[0021] Figure 3 It is a chromatogram for detecting a sample containing glycine palmitate dipeptide with methanol-water as the mobile phase;

[0022] Figure 4 It is a chromatogram for detecting a sample containing glycine palmitate dipeptide using an ultraviolet detector;

[0023] Figure 5 It is a chromatogram for the specificity investigation of the detection method for glycine palmitate dipeptide provided by the present invention;

[0024] Figure 6 It is a chromatogram for the sensitivity investigation of the detection method for glycine palmitate dipeptide provided by the present invention. Detailed Embodiments

[0025] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] The present invention provides a method for detecting palmitoyl glycine dipeptide. The liquid chromatography method is used to detect a sample containing palmitoyl glycine dipeptide. The mobile phase consists of an organic phase and an aqueous phase. The organic phase is a formic acid methanol solution, and the aqueous phase is a formic acid aqueous solution.

[0027] The sample containing palmitoyl glycine dipeptide can be the target product, intermediate product or raw material in the process of synthesizing the palmitoyl glycine dipeptide sample. The present invention has no special restrictions on the source of the sample containing palmitoyl glycine dipeptide.

[0028] Weigh a sample containing palmitoyl glycine dipeptide as the sample to be tested. In some possible implementation manners, dissolve the sample to be tested with methanol. After adding methanol to the sample to be tested, dissolve it at 30-70 °C, preferably at 40-60 °C. In some possible implementation manners, it is preferably assisted by ultrasonic, shaking or vortex for dissolution; after the dissolution is completed, it is preferably made up to volume with methanol, shaken well, and a sample solution to be tested is obtained. In some possible implementation manners, filter the sample solution to be tested, preferably filter it with an organic phase filter membrane. The specification of the organic phase filter membrane is 0.22-0.65 μm, preferably 0.45 μm. After filtration, the filtrate of the sample to be tested is obtained.

[0029] After the filtration is completed, perform liquid chromatography detection on the filtrate of the sample to be tested. The liquid chromatography detection conditions are as follows:

[0030] In the above liquid chromatography detection, the stationary phase of the chromatographic column used is C18, C16 or C30, preferably C18. The specification of the chromatographic column can be 4.6×250mm×5μm, 2.1×250mm×5μm or 10×250mm×5μm, preferably 4.6×250mm×5μm. The column temperature of the chromatographic column is 25-35 °C, preferably 30 °C. The present invention has no special restrictions on the source of the chromatographic column and its stationary phase, and it can be purchased on the market.

[0031] The mobile phase includes an organic phase and an aqueous phase. The organic phase is a formic acid methanol solution, in which the volume concentration of formic acid is 0.1-0.3%, preferably 0.1%; the aqueous phase is a formic acid aqueous solution, in which the volume concentration of formic acid is 0.1-0.3%, preferably 0.1%; the flow rate of the mobile phase is 1.0-1.4 mL / min, preferably 1.2 mL / min.

[0032] In the liquid chromatography detection, gradient elution is adopted, and the gradient elution program is shown in the following table:

[0033]

[0034] In the liquid chromatography detection, the injector temperature is 35 - 45 °C, preferably 40 °C; in some possible implementation manners, an electrospray detector (CAD detector) or an evaporative light scattering detector (ELSD detector) is used for detection, preferably the CAD detector is used for detection, the detector temperature is Low, the injection volume is 8 - 12 μL, preferably 10 μL.

[0035] The filtrate of the sample to be tested is detected under the above chromatographic detection conditions, and the purity of glycine palmitic acid dipeptide in the sample to be tested can be obtained by area normalization method.

[0036] The present invention also conducts methodological investigations on the detection method of glycine palmitic acid dipeptide provided, including system suitability investigation, precision investigation, specificity investigation and sensitivity investigation.

[0037] The present invention provides a detection method of glycine palmitic acid dipeptide, using a formic acid methanol solution as the organic phase and a formic acid aqueous solution as the aqueous phase, and using liquid chromatography to detect a sample containing glycine palmitic acid dipeptide. The detection method provided by the present invention can accurately detect glycine palmitic acid dipeptide. In addition, methodological investigations are conducted on the detection method provided by the present invention, and the experimental results show that the detection method of glycine palmitic acid dipeptide provided by the present invention has good system suitability, good precision, strong specificity and high sensitivity.

[0038] Example 1:

[0039] (1) Sample preparation

[0040] In this example, glycine palmitic acid dipeptide was synthesized by a liquid phase method from palmitoyl chloride and glycine under alkaline conditions. The synthesized target product was used as the sample to be tested. 100 mg of the sample to be tested was accurately weighed and placed in a 25 mL volumetric flask. An appropriate amount of methanol was added to dissolve the sample to be tested. At a temperature of 40 °C, ultrasonic treatment was carried out for 10 - 15 min. After the sample to be tested was dissolved, it was made up to the mark with methanol and shaken well. An appropriate amount of the sample solution to be tested was filtered through a 0.45 μm organic phase filter membrane to obtain the filtrate of the sample to be tested for detection.

[0041] (2) Instrument chromatographic conditions

[0042] The chromatographic column used was Thermo hypersil GOLD (4.6×250 mm×5 μm), the mobile phase was a 0.1% formic acid methanol solution - 0.1% formic acid aqueous solution system, and the gradient elution program was:

[0043]

[0044] During gradient elution, the pH value of the system is maintained constant. The flow rate of the mobile phase is 1.2 mL / min, the temperature of the injector is 40 °C, the column temperature of the chromatographic column is 30 °C, the detector is a CAD detector, the detector temperature is Low, and the injection volume is 10 μL.

[0045] (3) Sample detection

[0046] The filtrate of the sample to be measured is detected under the chromatographic conditions described in step (2), and a chromatogram as Figure 1 shown is obtained. It can be seen from Figure 1 that by using a formic acid methanol solution as the organic phase and a formic acid aqueous solution as the aqueous phase, palmitoyl glycine dipeptide can be successfully eluted and separated from the impurities in the sample to be measured. By calculation using the area normalization method, the purity of palmitoyl glycine dipeptide in the sample to be measured is 96.8%.

[0047] Example 2:

[0048] (1) Sample preparation

[0049] In this example, palmitoyl chloride and glycine were used as raw materials, and palmitoyl glycine dipeptide was synthesized by a liquid-phase method under alkaline conditions. The synthesized intermediate product was used as the sample to be measured. 100 mg of the sample to be measured was accurately weighed and placed in a 25 mL volumetric flask. An appropriate amount of methanol was added to dissolve the sample to be measured. At a temperature of 40 °C, it was sonicated for 10 - 15 min. After the sample to be measured was dissolved, it was made up to the mark with methanol and shaken well. An appropriate amount of the sample solution to be measured was filtered through a 0.45 μm organic phase filter membrane to obtain the filtrate of the sample to be measured for detection.

[0050] (2) Instrument chromatographic conditions

[0051] The chromatographic column used was Thermo hypersil GOLD (4.6×250 mm×5 μm), the mobile phase was a 0.1% formic acid methanol solution - 0.1% formic acid aqueous solution system, and the gradient elution program was:

[0052]

[0053] During gradient elution, the pH value of the system is maintained constant. The flow rate of the mobile phase is 1.2 mL / min, the temperature of the injector is 40 °C, the column temperature of the chromatographic column is 30 °C, the detector is a CAD detector, the detector temperature is Low, and the injection volume is 10 μL.

[0054] (3) Sample detection

[0055] The filtrate of the sample to be measured is detected under the chromatographic conditions described in step (2), and a chromatogram asFigure 2 The chromatogram shown is composed of Figure 2 As can be seen, by using a formic acid - methanol solution as the organic phase and a formic acid - aqueous solution as the aqueous phase, glycine palmitate dipeptide can be successfully eluted, and glycine palmitate dipeptide can be baseline - separated from the impurities in the sample to be tested, meeting the detection requirements. By calculation using the area normalization method, the purity of glycine palmitate dipeptide contained in the sample to be tested is 68.4%.

[0056] Comparative Example 1:

[0057] The sample to be tested in Example 1 was detected by the detection method in Example 1, but the mobile phase was changed to methanol - water. The detection results are shown in Figure 3 , and as can be seen from Figure 3 , when methanol - water is used as the mobile phase, glycine palmitate dipeptide co - elutes with impurity 1 (synthesis side - reaction product) in the sample to be tested and cannot be separated, failing to meet the detection requirements.

[0058] Comparative Example 2:

[0059] The sample to be tested in Example 1 was detected by the detection method in Example 1, but the detector was changed to an ultraviolet detector. The detection results are shown in Figure 4 , and as can be seen from Figure 4 , when detected by an ultraviolet detector, since impurity 2 (synthesis raw material) has no ultraviolet absorption, this impurity cannot be effectively detected, failing to meet the detection requirements.

[0060] Example 3:

[0061] Methodology investigation was carried out on the detection method of glycine palmitate dipeptide provided by the present invention.

[0062] (1) System suitability investigation

[0063] Taking glycine palmitate dipeptide synthesized by the same batch of liquid - phase method as the sample, preparing 1 test solution, and injecting samples continuously for 6 times by the detection method provided in Example 1. The calculation results are shown in Table 1. As can be seen from Table 1, for the detection of peak area, the relative standard deviation of the experimental results is small, indicating that the system suitability of the detection method of glycine palmitate dipeptide provided by the present invention is good.

[0064] Table 1

[0065]

[0066] (2) Precision investigation

[0067] Take the palmitoyl glycine dipeptide synthesized by the same batch of liquid phase method as the sample, prepare 6 test solutions, inject each test solution with 1 injection using the detection method provided in Example 1, and the calculation results are shown in Table 2. It can be seen from Table 2 that for the purity detection, the relative standard deviation of the detection results is small, indicating that the precision of the detection method for palmitoyl glycine dipeptide provided by the present invention is good.

[0068] Table 2

[0069]

[0070] (3) Specificity investigation

[0071] Prepare a mixed solution of palmitoyl glycine dipeptide sample, impurity 1 (synthesis side reaction product) and impurity 2 (synthesis raw material), and analyze it using the detection method provided in Example 1. The resolution is greater than 2.0, and the chromatogram is as Figure 5 shown. It can be seen from Figure 5 that the palmitoyl glycine dipeptide sample, impurity 1 (synthesis side reaction product) and impurity 2 (synthesis raw material) can achieve baseline separation, indicating that the detection method for palmitoyl glycine dipeptide provided by the present invention has strong specificity.

[0072] (4) Sensitivity investigation

[0073] Adopt the method of stepwise dilution, and analyze palmitoyl glycine dipeptide solutions with different concentrations using the detection method provided in Example 1. When the signal-to-noise ratio is not less than 10, the measured lowest quantitative detection concentration is only 0.1 mg / g, indicating that the detection method for palmitoyl glycine dipeptide provided by the present invention has good sensitivity. The chromatogram is as Figure 6 shown.

[0074] The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A detection method for glycine palmitic acid dipeptide, characterized in that, The sample containing glycine palmitate dipeptide is detected by liquid chromatography, and the mobile phase includes formic acid methanol solution and formic acid aqueous solution; gradient elution is adopted during the detection process; the volume concentration of formic acid in the formic acid methanol solution is 0.1-0.3%, and the volume concentration of formic acid in the formic acid aqueous solution is 0.1-0.3%; The gradient elution program is as follows:

2. The detection method according to claim 1, characterized in that, The flow rate of the mobile phase is 1.0-1.4 mL / min.

3. The detection method according to claim 1, characterized in that, It also includes dissolving the sample containing glycine palmitate dipeptide with methanol.

4. The detection method according to claim 3, characterized in that, The dissolution is carried out at a temperature of 30-70 °C.

5. The detection method according to claim 1, characterized in that, During the detection process, the stationary phase of the chromatographic column used is C18, C16 or C30.

6. The detection method according to claim 5, characterized in that, The column temperature of the chromatographic column is 25-35 °C.

7. The detection method according to claim 1, characterized in that, During the detection process, an electrospray detector or an evaporative light scattering detector is used for detection.

8. The detection method according to claim 1, characterized in that, During the detection process, the injector temperature is 35-45 °C, and the injection volume is 8-12 μL.

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