A method for separating and analyzing enantiomers in acetyl tetrapeptide-3 and application thereof

Abstract

The application discloses a separation and analysis method of enantiomers in acetyl tetrapeptide-3 and application thereof. The application adopts high performance liquid chromatography for separation and analysis, wherein a hydrophilic octadecylsilane bonded silica gel column is selected, and an organic acid solution with a concentration of 0.05-0.15% by volume is used to elute at a flow rate of 0.2-1.0 mL / min, so that acetyl tetrapeptide-3 and enantiomers thereof can be effectively separated and analyzed, and the separation degree reaches 1.5 or above. The application avoids using organic reagents, is environment-friendly and less toxic to human bodies; and the application avoids configuring special instruments, is simple to operate and low in cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of preparation and detection analysis, and particularly relates to a separation and analysis method of enantiomers in acetyl tetrapeptide-3 and application thereof. BACKGROUND

[0002] Since most of the amino acids are chiral molecules (except glycine), the generation of enantiomeric impurities is inevitable in the process of polypeptide synthesis, or introduced from the raw material of amino acid, which may lead to changes in biological activity. Therefore, the enantiomeric impurities in polypeptide synthesis should be controlled.

[0003] For the detection of enantiomeric impurities of amino acids, shorter polypeptides, and chemical drugs, the following methods are usually used: normal phase chiral column method, pre-column or post-column derivatization method, and mobile phase chiral additive method. The normal phase chiral column method usually has the disadvantages of long time consumption, low sensitivity, complex operation, high toxicity, and the need for special instruments. The pre-column or post-column derivatization method has high requirements for experimental operation and reagents, and the derivative products are not single. The separation effect of the mobile phase chiral additive method is unstable, the service life of the chromatographic column is short, and the sensitivity is very low. If some cosmetic polypeptide enantiomeric impurities can be separated by reverse phase chromatography, organic reagents such as methanol and acetonitrile will be used, which have certain toxicity.

[0004] Acetyl tetrapeptide-3 is a cosmetic polypeptide containing 4 amino acids, and the amino acid sequence is Ac-Lys-Gly-His-Lys-NH2, the CAS number is 827306-88-7, and it has the effect of promoting hair growth.

[0005] There is no separation and analysis method of enantiomers in acetyl tetrapeptide-3 in the prior art. SUMMARY

[0006] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a separation and analysis method of enantiomers in acetyl tetrapeptide-3.

[0007] Another purpose of the present application is to provide the application of the above-mentioned separation and analysis method of enantiomers in acetyl tetrapeptide-3.

[0008] The purpose of the present application is achieved by the following technical scheme: a separation and analysis method of enantiomers in acetyl tetrapeptide-3, comprising the following steps: using high performance liquid chromatography for separation and analysis, and the chromatographic conditions include the following parameters:

[0009] Chromatographic column: hydrophilic octadecylsilane bonded silica gel column;

[0010] Mobile phase: organic acid solution with a concentration of 0.05-0.15% (volume percent).

[0011] The hydrophilic octadecylsilane bonded silica gel column is preferably an Ultimate ALK C18 column.

[0012] The organic acid is preferably one or more of formic acid, acetic acid and phosphoric acid.

[0013] The concentration of the organic acid solution is preferably 0.1% by volume.

[0014] The elution in the chromatographic conditions is isocratic elution.

[0015] The time of the isocratic elution is preferably 10 min or more; more preferably 20 min.

[0016] The flow rate of the elution is preferably 0.2-1.0 mL / min; more preferably 0.5-1.0 mL / min.

[0017] The chromatographic conditions further comprise the following conditions: detection wavelength 200-220 nm; column temperature 20-30°C.

[0018] The detection wavelength is preferably 210 nm.

[0019] The column temperature is preferably 25°C.

[0020] The chromatographic conditions further comprise: injection volume 5 μL.

[0021] The above method for separating and / or analyzing the enantiomers of acetyl tetrapeptide-3 is used for separating and / or analyzing the enantiomers of acetyl tetrapeptide-3 in an acetyl tetrapeptide-3 sample.

[0022] The present application has the following advantages and effects relative to the prior art:

[0023] 1. The present application first separates and analyzes acetyl tetrapeptide-3 and its enantiomers, and the separation degree is 1.5 or more;

[0024] 2. The present application avoids using organic reagents, and is environmentally friendly and less toxic to the human body.

[0025] 3. The present application avoids the use of special instruments, and is simple to operate and low in cost. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a liquid chromatogram of acetyl tetrapeptide-3 of Example 1.

[0027] Figure 2 is a liquid chromatogram of the enantiomers of acetyl tetrapeptide-3 of Example 1.

[0028] Figure 3 is a liquid chromatogram of the mixed standard verification solution of Example 1.

[0029] Figure 4 Liquid chromatogram of acetyl tetrapeptide-3 of Comparative Example 1.

[0030] Figure 5 Liquid chromatogram of acetyl tetrapeptide-3 enantiomer of Comparative Example 1.

[0031] Figure 6 Liquid chromatogram of mixed solution of Comparative Example 1.

[0032] Figure 7 Liquid chromatogram of acetyl tetrapeptide-3 of Comparative Example 2.

[0033] Figure 8 Liquid chromatogram of acetyl tetrapeptide-3 enantiomer of Comparative Example 2.

[0034] Figure 9 Liquid chromatogram of acetyl tetrapeptide-3 of Comparative Example 2 superimposed with acetyl tetrapeptide-3 enantiomer of Comparative Example 2.

[0035] Figure 10 Liquid chromatogram of acetyl tetrapeptide-3 of Comparative Example 3.

[0036] Figure 11 Liquid chromatogram of acetyl tetrapeptide-3 enantiomer of Comparative Example 3.

[0037] Figure 12 Liquid chromatogram of mixed sample of Comparative Example 3. DETAILED DESCRIPTION

[0038] The present application will be further described in conjunction with the following examples and drawings, but the embodiments of the present application are not limited thereto.

[0039] Example 1

[0040] 1. Instruments and reagents: Shimadzu LC-2010CHT high performance liquid chromatograph; formic acid (Sigma-Aldrich).

[0041] Samples: acetyl tetrapeptide-3 sample (Hefei Guopei Biotechnology Co., Ltd.), acetyl tetrapeptide-3 enantiomer (Hefei Guopei Biotechnology Co., Ltd.).

[0042] 2. Experimental method:

[0043] 2.1 Preparation of samples: Take the acetyl tetrapeptide-3 sample, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with water to prepare an acetyl tetrapeptide-3 sample solution of about 1 mg / mL. Take the acetyl tetrapeptide-3 enantiomer, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with water to prepare an acetyl tetrapeptide-3 enantiomer solution of about 1 mg / mL. Take 1 mL of the acetyl tetrapeptide-3 sample solution and 1 mL of the acetyl tetrapeptide-3 enantiomer solution, mix them evenly in equal volumes to prepare a mixed sample verification solution, which is ready for use. Each sample is filtered through a 0.22 μm filter before injection.

[0044] 2.2 Chromatographic conditions: Use a Shimadzu LC-2010CHT high performance liquid chromatograph, the chromatographic column is an Ultimate ALK C18 5 μm 4.6 x 250 mm; the mobile phase is 0.1% (v / v) formic acid aqueous solution; the flow rate is 0.5 mL / min; the detection wavelength is 210 nm; the column temperature is 25°C; the injection volume is 5 μL; and the isocratic elution time is 20 min.

[0045] 3. Experimental results:

[0046] The integration results of the chromatogram of the acetyl tetrapeptide-3 sample are shown in Table 1, and the chromatogram is shown in Figure 1 .

[0047] Table 1 Integration results of acetyl tetrapeptide-3 sample

[0048]

[0049] The integration results of the chromatogram of the acetyl tetrapeptide-3 enantiomer are shown in Table 2, and the chromatogram is shown in Figure 2 .

[0050] Table 2 Integration results of acetyl tetrapeptide-3 enantiomer

[0051]

[0052] The integration results of the chromatogram of the mixed sample verification solution are shown in Table 3, and the chromatogram is shown in Figure 3 .

[0053] Table 3

[0054]

[0055] From the above results, it can be seen that the retention time of acetyl tetrapeptide-3 is 7.843 min (as shown in Figure 1 ); the retention time of acetyl tetrapeptide-3 enantiomer is 9.626 min (as shown in Figure 2 ); and in the chromatogram of the mixed sample verification solution, the two are separated to the baseline after mixing, with a separation degree of 3.509, and the separation effect is very good (as shown in Figure 3(As shown).

[0056] Comparative Example 1

[0057] 1. Instruments and reagents: Shimadzu LC-2010CHT high performance liquid chromatograph, with Chameleon 7 network version chromatography workstation as the control software; potassium dihydrogen phosphate, sodium 1-pentanesulfonate, phosphoric acid, acetonitrile.

[0058] Samples: Acetyl tetrapeptide-3 sample (Hefei Guotai Biotechnology Co., Ltd.), Acetyl tetrapeptide-3 (Hefei Guotai Biotechnology Co., Ltd.).

[0059] 2. Experimental Methods:

[0060] 2.1 Sample Preparation: Take an appropriate amount of acetyl tetrapeptide-3 sample, place it in a 20 mL volumetric flask, dissolve and dilute with water to the mark to prepare an acetyl tetrapeptide-3 sample solution of approximately 1 mg / mL. Take an appropriate amount of acetyl tetrapeptide-3 enantiomer, place it in a 20 mL volumetric flask, dissolve and dilute with water to the mark to prepare an acetyl tetrapeptide-3 enantiomer solution of approximately 1 mg / mL. Take 1 mL each of the acetyl tetrapeptide-3 sample solution and the acetyl tetrapeptide-3 enantiomer solution, mix them evenly with equal volumes to prepare a mixed sample solution for later use. All samples were filtered through a 0.22 μm filter membrane before injection.

[0061] 2.2 Chromatographic conditions: A Shimadzu LC-2010CHT high-performance liquid chromatograph was used, with an XAqua C18 5μm 4.6×250mm reversed-phase column. Mobile phase A was 25 mmol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.6 with phosphoric acid), containing 5 mmol / L sodium 1-pentanesulfonate; mobile phase B was 0.1% (v / v) phosphoric acid aqueous solution-acetonitrile (volume ratio 20:80). The preparation method of mobile phase A was as follows: Weigh 1.74 g of sodium 1-pentanesulfonate and 6.80 g of potassium dihydrogen phosphate, add deionized water to a final volume of 2 L, dissolve completely, adjust the pH to 2.60 with phosphoric acid, filter, and sonicate. The preparation method of mobile phase B was as follows: Measure 500 mL of deionized water, add 0.5 mL of phosphoric acid, mix well, filter, take 400 mL of the filtrate, add 1600 mL of acetonitrile, mix well, and set aside. The flow rate was 1.0 mL / min; the detection wavelength was 210 nm; the column temperature was 35 °C; and the injection volume was 5 μL. The elution gradient was as follows:

[0062] Table 4

[0063] Time Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 8 95 5 15 80 20 30 0 100 40 100 0 50 100 0

[0064] 3. Experimental Results:

[0065] like Figure 4 As shown, the retention time of the acetyl tetrapeptide-3 sample was 15.990 min;Figure 5 As shown in the figure, the retention time of the acetyl tetrapeptide-3 enantiomer is 16.143 min; as shown in the figure, Figure 6 As shown in the figure, the separation effect of the mixed sample solution is poor.

[0066] Comparative Example 2 Comparison of different types of chromatographic columns

[0067] 1. Instruments and reagents: Agilent 1260 high performance liquid chromatograph, control software is chameleon 7 network version chromatographic workstation; phosphoric acid, acetonitrile.

[0068] Sample: Acetyl tetrapeptide-3 sample (Hefei Guopei Biological Technology Co., Ltd.), acetyl tetrapeptide-3 enantiomer (Hefei Guopei Biological Technology Co., Ltd.).

[0069] 2. Experimental method:

[0070] 2.1 Preparation of sample: Take acetyl tetrapeptide-3 sample, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with water to prepare about 1 mg / mL acetyl tetrapeptide-3 sample solution. Take acetyl tetrapeptide-3 enantiomer, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with water to prepare about 1 mg / mL acetyl tetrapeptide-3 enantiomer solution. Each sample is filtered through a 0.22 μm filter membrane before injection.

[0071] 2.2 Chromatographic conditions: Agilent 1260 high performance liquid chromatograph, chromatographic column is Ultimate XB-C18 5 μm 4.6 x 250 mm reversed phase chromatographic column. The mobile phase A is 0.1% (v / v) phosphoric acid aqueous solution: acetonitrile = 96:4 (v / v) mixed to obtain; the mobile phase B is 0.1% (v / v) phosphoric acid aqueous solution: acetonitrile = 20:80 (v / v) mixed to obtain. The flow rate is 1.0 mL / min; the detection wavelength is 210 nm, the column temperature is 30℃, the injection amount is 5 μL. The elution gradient is as follows:

[0072] Table 5

[0073] Time Mobile phase A (%) Mobile phase B (%) 0 100 0 15 85 15 31 0 100 35 0 100 35.1 100 0 50 100 0

[0074] 3. Experimental results:

[0075] As shown in the figure, the retention time of the acetyl tetrapeptide-3 sample is 15.938 min; as shown in the figure, Figure 7 As shown in the figure, the retention time of the acetyl tetrapeptide-3 enantiomer is 16.115 min; Figure 8 As shown in the figure, the separation effect is poor. Figure 7 And Figure 8 Superimposed comparison as shown in the figure, Figure 9 As shown in the figure, the separation effect is poor.

[0076] Comparative Example 3 Comparison of different hydrophilic chromatographic columns

[0077] 1. Instruments and reagents: Waters ACQUITY UPLC H-CLASS ultra-high performance liquid chromatograph, control software Empower3 chromatography workstation; formic acid, acetonitrile.

[0078] Sample: Acetyl tetrapeptide-3 sample (Hefei Guopei Biological Technology Co., Ltd.), acetyl tetrapeptide-3 enantiomer (Hefei Guopei Biological Technology Co., Ltd.).

[0079] 2. Experimental method:

[0080] 2.1 Preparation of sample: Take acetyl tetrapeptide-3 sample, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with water to prepare about 1 mg / mL acetyl tetrapeptide-3 sample solution. Take acetyl tetrapeptide-3 enantiomer, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with water to prepare about 1 mg / mL acetyl tetrapeptide-3 enantiomer solution. Take 1 mL of acetyl tetrapeptide-3 sample solution and 1 mL of acetyl tetrapeptide-3 enantiomer solution, mix them evenly in equal volume as the mixed sample solution, ready for use. Each sample is filtered through a 0.22 μm filter membrane before injection.

[0081] 2.2 Chromatographic conditions: Waters ACQUITY UPLC H-CLASS ultra-high performance liquid chromatograph was used, and the chromatographic column was Thermo Accucore aQ C18 1.7 μm 2.1 x 100 mm reversed-phase chromatographic column. The mobile phase A was 0.1% (v / v) formic acid aqueous solution; the mobile phase B was pure acetonitrile. The flow rate was 0.2 mL / min; the detection wavelength was 210 nm, the column temperature was 30 ℃, and the injection volume was 0.1 μL. The elution gradient was as follows:

[0082] Table 6

[0083] Time Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 15 60 40 20 100 0 25 100 0

[0084] 3. Experimental results:

[0085] The retention of acetyl tetrapeptide-3 sample and acetyl tetrapeptide-3 enantiomer was very weak, among which the retention time of acetyl tetrapeptide-3 sample was 0.414 min, as shown in Figure 10 The retention time of acetyl tetrapeptide-3 enantiomer was 0.412 min, as shown in Figure 11 The retention time of the mixed sample solution was 0.412 min, and acetyl tetrapeptide-3 and its enantiomer could not be separated, as shown in Figure 12

[0086] ​The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A method for separating and analyzing enantiomers in acetyl tetrapeptide-3, characterized by It comprises the following steps: separation and analysis by high performance liquid chromatography, and the chromatographic conditions comprise the following parameters: The chromatographic column is a hydrophilic octadecylsilane bonded silica gel column; The mobile phase is an organic acid solution with a concentration of 0.05-0.15% (volume percent); The hydrophilic octadecylsilane bonded silica gel column is an Ultimate ALK C18 chromatographic column; The organic acid is formic acid; The elution in the chromatographic conditions is isocratic elution.

2. The separation and analysis method of enantiomers in acetyl tetrapeptide-3 according to claim 1, characterized in that: The concentration of the organic acid solution is 0.1% (volume percent).

3. The separation and analysis method of enantiomers in acetyl tetrapeptide-3 according to claim 1, characterized in that: The flow rate of the elution is 0.2-1.0 mL / min; The isocratic elution time is more than 10 min.

4. The separation and analysis method of enantiomers in acetyl tetrapeptide-3 according to claim 3, characterized in that: The flow rate of the elution is 0.5-1.0 mL / min; The isocratic elution time is 20 min.

5. The separation and analysis method of enantiomers in acetyl tetrapeptide-3 according to claim 1, characterized in that: The chromatographic conditions further comprise the following conditions: detection wavelength 200-220 nm; column temperature 20-30℃.

6. The separation and analysis method of enantiomers in acetyl tetrapeptide-3 according to claim 5, characterized in that: The detection wavelength is 210 nm; The column temperature is 25℃.

7. The use of the separation and analysis method of enantiomers in acetyl tetrapeptide-3 according to any one of claims 1-6 in separating and / or analyzing acetyl tetrapeptide-3 enantiomers in an acetyl tetrapeptide-3 sample.

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