CN115267013B

CN115267013B - HPLC analysis method suitable for various palmitoyl peptides

Info

Publication number: CN115267013B
Application number: CN202211171513.XA
Authority: CN
Inventors: 王海梅; 张志乾; 王嘉鹏; 吴奕瑞; 傅伟; 朱家平; 江翱; 邱�益; 王帆; 崔华; 谭洪群
Original assignee: Shandong Dry Peptide Biotechnology Co ltd; Guangzhou Qianxiang Biotechnology Co Ltd
Current assignee: Shandong Dry Peptide Biotechnology Co ltd; Guangzhou Qianxiang Biotechnology Co Ltd
Priority date: 2022-09-26
Filing date: 2022-09-26
Publication date: 2022-12-13
Anticipated expiration: 2042-09-26
Also published as: CN115267013A

Abstract

The invention relates to an HPLC analysis method suitable for various palmitoyl peptides, which is characterized by comprising the following steps: the first step is as follows: preparing a solution of one or more palmitoyl peptide samples to be tested, wherein the palmitoyl peptide samples can be a single palmitoyl peptide or a mixture of palmitoyl peptides; the second step is that: setting liquid phase chromatographic conditions according to the type of the prepared to-be-detected palmitoyl peptide sample; the third step: preparing a mobile phase for analysis; the fourth step: determining one of the palmitoyl peptide samples to be determined by adopting an analysis program; the fifth step: cleaning the chromatographic column for 1 to 5min by using a mobile phase B; and a sixth step: and repeating the fourth step and the fifth step, and sequentially determining other undetected palmitoyl peptide samples to be detected. The HPLC analysis method suitable for the plurality of palmitoyl peptides is simple to operate, saves a large amount of time cost and economic cost when continuously separating and determining the plurality of palmitoyl peptides, and has universality on the plurality of palmitoyl peptides.

Description

HPLC analysis method suitable for various palmitoyl peptides

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a High Performance Liquid Chromatography (HPLC) analysis method suitable for various palmitoyl peptides.

Background

In the prior art, many reports have been made on HPLC analysis methods for peptides, however, few reports have been made on the analysis method for palmitoyl peptides. The palmitoyl peptide is modified by one more palmitoyl group than a normal peptide, so that the properties of the peptide are greatly changed, and the commonly used peptide analysis method is not suitable for the palmitoyl peptide. The prior art document CN 106546673A discloses a method for separating palmitoyl pentapeptide-3 by high performance liquid chromatography, however, in order to improve the peak shape and separation effect of chromatographic peak, trifluoroacetic acid is added into the mobile phase. This results in the end product having acid or trifluoroacetic acid residues which make the product acidic and noticeably sour, while some peptides are actually unstable under acidic conditions, affecting its shelf life. Furthermore, trifluoroacetic acid is a strong acid, which has high requirements on the tolerance of chromatographic columns, and a general chromatographic column cannot bear the strong acid, so that an enhanced chromatographic column is required.

In addition, it is also rare in the prior art to use a common HPLC method for continuous separation and determination of various peptides. Generally, each peptide corresponds to a specific method, so that the preparation work of the mobile phase is complicated, the variety of required reagents is various, and great investment is required in both management and required space. For example: the mobile phase for peptide 1 was methanol and water and the analytical procedure used was procedure 1, the column used was a silica gel column, the analysis time was 10min; whereas the mobile phase for peptide 2 was acetonitrile and water, the analytical procedure used was procedure 2, the column used was an octadecyl column and the analysis time was 10min. When the analysis of peptide 1 was completed and the analysis of peptide 2 was switched, it took about 2 hours to clean the silica gel column, then to change it to the octadecyl column, to prepare acetonitrile and water mobile phase, to clean the octadecyl column in the early stage, and to balance it. However, the analysis time is only 10min, which results in a large waste of time and cost. If the method can be unified, 12 samples can be analyzed in 2 hours of wasted time due to the replacement method.

Related documents have reported that: different peptides use different mobile phases, such as: the acetonitrile or methanol is mixed with water to achieve the separation purpose. There are also reports of the use of pure organic reagents or inorganic salts. However, these methods are only suitable for one kind of peptide or protein, are not generally used for most of peptides or proteins, and are very easy to cause blockage of instruments or chromatographic columns if the washing is incomplete when inorganic salts are used, thereby causing troubles to subsequent work.

In the past work, the situation that several peptides cannot be completely separated easily occurs in peptide analysis, and the complete separation is required in peptide content analysis, otherwise, the content of the peptides cannot be accurately calculated. Meanwhile, when analyzing a plurality of peptides, one method is usually corresponding to each peptide, and the method comprises an analysis program, a mobile phase component and a proportion. Making the operation complicated.

The inventors of the present invention have conducted extensive studies for a long time and have not found any report of an HPLC analysis method capable of continuously analyzing a plurality of palmitoyl peptides.

Therefore, there is a need to search for a more versatile high performance liquid chromatography method with general applicability to a wider variety of palmitoyl peptides.

Disclosure of Invention

Problems to be solved by the invention

Aiming at the problems in the prior art, one of the purposes of the invention is to solve the problem of time cost waste caused by the procedures of replacing or cleaning a chromatographic column, replacing a mobile phase, rebalancing and the like when a plurality of palmitoyl peptide samples are continuously subjected to HPLC method separation and measurement.

Still another object of the present invention is to solve the problems of incomplete washing, easy blockage of the apparatus or chromatographic column, etc., when the mobile phase contains inorganic salts.

The most important object of the present invention is to solve the problem of palmitoyl peptide dissolution. In addition, another object of the present invention is to solve the problem of the prior art that trifluoroacetic acid added for improving the peak shape of chromatographic peak is burdened on the chromatographic column.

Means for solving the problems

The present invention relates to:

1. an HPLC analysis method suitable for various palmitoyl peptides, which is characterized by comprising the following steps:

the first step is as follows: preparing a solution of one or more palmitoyl peptide samples to be tested, wherein the palmitoyl peptide samples can be a single palmitoyl peptide or a mixture of palmitoyl peptides;

the second step is that: setting liquid phase chromatographic conditions according to the type of the prepared to-be-detected palmitoyl peptide sample;

the third step: preparing a mobile phase for analysis;

the fourth step: determining one of the palmitoyl peptide samples to be determined by adopting an analysis program;

the fifth step: cleaning the chromatographic column for 1 to 5min by using a mobile phase B;

and a sixth step: repeating the fourth step and the fifth step, and sequentially measuring other undetected palmitoyl peptide samples to be measured;

wherein:

for the mobile phase for analysis, mobile phase a was 0.2% phosphoric acid aqueous solution, and pH was adjusted to 3.5 with triethylamine; the mobile phase B is methanol;

the analytical procedure was isocratic elution, elution concentration: mobile phase A:15%, mobile phase B:85 percent.

2. The HPLC analysis method for various palmitoyl peptides according to item 1, characterized in that: the palmitoyl peptide sample to be detected is prepared according to the following proportion, 0.008 to 0.012g of palmitoyl peptide is weighed, 0 to 8.5mL of methanol is added according to needs, 0.2mL of phosphoric acid is added, the palmitoyl peptide is dissolved and then is supplemented to 10mL by adding ultrapure water, and the solution of the palmitoyl peptide sample to be detected with the concentration of 0.0008 to 0.0012g/mL is obtained after dissolution.

3. The HPLC analysis method for various palmitoyl peptides according to item 2, characterized in that: the concentration of the palmitoyl peptide sample to be detected is 0.001g/ml.

4. The HPLC analysis method for various palmitoyl peptides according to item 1 or 2, characterized in that: the wavelength range is 200 to 300nm.

5. The HPLC analysis method for various palmitoyl peptides according to item 1 or 2, characterized in that: the chromatographic column is a C18 packing chromatographic column, and the flow rate is 1mL/min.

6. The HPLC analysis method for various palmitoyl peptides according to item 1 or 2, characterized in that: the temperature of the chromatographic column is room temperature, the sample injection amount is 8 to 15 mu L, the temperature of the sample injector is ambient temperature, and the collection time of the sample to be detected is 10 to 30min.

7. The HPLC analysis method for various palmitoyl peptides according to item 1 or 2, characterized in that: the palmitoyl peptide is palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl tripeptide-8, palmitoyl tetrapeptide-7, and palmitoyl pentapeptide-4.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention discloses a novel HPLC analysis method suitable for various palmitoyl peptides, which can realize continuous separation and determination of various palmitoyl peptides, and can realize the aim of improving the peak shape without adding trifluoroacetic acid, but does not have the adverse effects that strong acids such as trifluoroacetic acid are not beneficial to the tolerance of a chromatographic column or a reinforced chromatographic column with higher performance is needed, and the adverse effects that trifluoroacetic acid can influence the storage and efficacy of some peptides.

Secondly, the HPLC analysis method for various palmitoyl peptides integrates the analysis methods for various palmitoyl peptides, and reduces the consumption of manpower and material resources during analysis. The chromatographic column does not need to be replaced during analysis of different palmitoyl peptides, only the chromatographic column needs to be cleaned, the analysis program does not need to be replaced, the detection wavelength does not need to be changed, the temperature of the chromatographic column does not need to be changed, the mobile phase does not need to be prepared again, the degassing does not need to be carried out again, and analysis and purity calculation of various palmitoyl peptides can be realized. And the method is used for analyzing the palmitoyl peptide with different concentrations, so that good linearity can be obtained.

The HPLC analysis method suitable for the plurality of palmitoyl peptides is simple to operate, saves a large amount of time cost and economic cost when continuously separating and determining the plurality of palmitoyl peptides, and greatly simplifies the complexity of operation. The technology can greatly save the time for preparing the mobile phase and the waiting time consumed during switching peptide analysis, not only can save the time cost, but also can save the space occupied by mobile phase storage and chromatographic bottle storage and the labor consumption in the process of preparation and switching, thereby being more beneficial to the realization of whole-process automation by utilizing the method.

Drawings

FIG. 1 is an HPLC chromatogram of palmitoyl tripeptide-1 from example 1.

FIG. 2 is an HPLC chromatogram of palmitoyl tripeptide-5 from example 2.

FIG. 3 is an HPLC chromatogram of palmitoyl tripeptide-8 from example 3.

FIG. 4 is an HPLC chromatogram of palmitoyl tetrapeptide-7 in example 4.

FIG. 5 is an HPLC chromatogram of palmitoyl pentapeptide-4 in example 5.

FIG. 6 is an HPLC chromatogram of palmitoyl tripeptide-5 of comparative example 1.

FIG. 7 is an HPLC chromatogram of palmitoyl pentapeptide-4 of comparative example 2.

Detailed Description

The HPLC analysis method applicable to various palmitoyl peptides comprises the following steps:

the third step: preparing a mobile phase for analysis;

and a sixth step: and repeating the fourth step and the fifth step, and sequentially determining other undetected palmitoyl peptide samples to be detected.

The palmitoyl peptide may be, but is not limited to, for example, palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl tripeptide-8, palmitoyl tetrapeptide-7, palmitoyl pentapeptide-4, and the like.

The mobile phase is most preferably phase A:0.2% phosphoric acid aqueous solution, adjusting pH to 3.5 with triethylamine; phase B: methanol, HPLC grade. The range is selected to obtain the best separation. Although the separation effect can be achieved by the mobile phase in other ranges, the separation effect and the separation efficiency are inferior to those achieved by the mobile phase.

In order to enable the peak of the target peptide to be completely displayed, the concentration of the peptide is not high enough, otherwise, the target peak is capped, and therefore, even if a mixed peak appears at the peak top, the judgment cannot be carried out; but the concentration cannot be too low, otherwise even if there is a stray peak, the peak height is too low and the baseline is misjudged to be uneven. The concentrations of the palmitoyl peptide samples to be tested were therefore: the concentration of the palmitoyl peptide sample to be detected is 0.0008 to 0.0012g/ml, and preferably 0.001g/ml.

The palmitoyl peptide is different from the common short peptide because the structure of the palmitoyl peptide is modified by one more palmitoyl group (hexadecanoyl group) compared with the common peptide, so that the property of the peptide is greatly changed, namely the change from water solubility to fat solubility is realized, but the few peptides have stronger hydrophilicity than the hydrophobicity of the palmitoyl group because the amino acid of the peptide is hydrophilic, and the palmitoyl group still shows water solubility even if the palmitoyl group is carried, such as palmitoyl tripeptide-5. HPLC requires complete dissolution of palmitoyl peptide for detection, but most palmitoyl peptide cannot be dissolved in water, and the solvent selection needs to consider that the peptide can be completely dissolved and the analysis of the peptide cannot be influenced by the selected solvent, namely, the light absorption of the solvent and the light absorption of the peptide cannot be overlapped, and the solvent cannot corrode an HPLC analysis system. Therefore, the formulation of palmitoyl peptide samples becomes particularly important for HPLC analysis.

The palmitoyl peptide sample to be detected is prepared according to the following proportion: weighing 0.008 to 0.012g of palmitoyl peptide, adding 0 to 8.5mL of methanol according to needs, adding 0.2mL of phosphoric acid, dissolving the palmitoyl peptide, and adding ultrapure water to supplement the dissolved palmitoyl peptide to 10mL to obtain a 0.1% peptide solution. Weighing 0.01g of palmitoyl tripeptide, dissolving in 10mL of ultrapure water to obtain a solution of the palmitoyl peptide sample to be detected, wherein the concentration of the palmitoyl tripeptide is 0.0008 to 0.0012g/mL.

The detection wavelength is determined by the nature of the peptide to be detected and is not influenced by the analysis conditions. However, the wavelength range is preferably 200 to 300nm, more preferably 210 to 230nm, still more preferably 260 to 280nm, and particularly preferably 215nm. The reason is that: when a full-wavelength scan is performed on all palmitoyl peptides, most palmitoyl peptides have strong light absorption at 215nm, so 215nm is most preferably used as the detection wavelength of HPLC analysis.

The column is a conventionally used column, preferably a column packed with C18, such as AgilentZORBAX SB-C18.

Isocratic elution is adopted, and the elution concentration is as follows: 15% of phase A; 85% of phase B.

The flow rate is generally set to 0.3 to 5mL/min, preferably 1mL/min.

The column temperature of the chromatographic column is 25 ℃, and the sample amount is 10-15 mu L, preferably 10 mu L.

The temperature of the sample injector is the ambient temperature, and the collection time of the sample to be detected is 10 to 30min, preferably 15min.

The technical solution of the present invention is further described below by means of specific examples.

The invention is further illustrated by the following examples, but not by way of limitation, in connection with the accompanying drawings. It is to be understood, however, that these examples are illustrative only and are not intended to limit the present invention. Unless otherwise specified, the raw materials used in the examples of the present invention are all those commonly used in the art, and the methods used in the examples are all those conventional in the art.

Examples

Instruments and conditions:

an Agilent1260InfinityII LC high performance liquid chromatograph and an OpenLabCDS2 software system are adopted; taking an agent ZORBAX SB-C18 (250 multiplied by 4.6 mm) as a separation column, wherein the column temperature is 25 ℃; the ultraviolet detection wavelength is 215nm.

The experimental steps are as follows:

0.01g of palmitoyl tripeptide-1 (example 1), palmitoyl tripeptide-5 (example 2, comparative example 1), palmitoyl tripeptide-8 (example 3), palmitoyl tetrapeptide-7 (example 4), and palmitoyl pentapeptide-4 (example 5, comparative example 2) were weighed, respectively. The weighed palmitoyl tripeptide-1 (example 1), palmitoyl tripeptide-8 (example 3), palmitoyl tetrapeptide-7 (example 4) and palmitoyl pentapeptide-4 (example 5) were dissolved in 8.5mL of methanol and then 0.2mL of phosphoric acid, and the mixture was dissolved and then 10mL of ultrapure water was added to make up a 0.1% peptide solution. 0.01g of weighed palmitoyl tripeptide-5 is dissolved in 10mL of ultrapure water to obtain a 0.1% palmitoyl tripeptide-5 solution. The five peptides are all products from Biotech (Guangzhou) GmbH, which are verified by Peking Baishipaike Biotech GmbH to have peptide sequences and molecular weights.

Mobile phase: a:0.2% phosphoric acid aqueous solution, adjusting pH to 3.5 with triethylamine; b methanol, HPLC grade.

Elution concentration: 15% of phase A; 85% of phase B.

Flow rate: 1.0mL/min.

The temperature was 25 ℃.

Sample introduction amount: 10uL.

And (3) analysis program: isocratic elution was performed using the elution concentrations described above.

The high performance liquid chromatography analyses of examples 1 to 5 were continuously carried out under the above-mentioned chromatographic conditions, and chromatograms were recorded, and when the test samples were replaced for the test, operations such as replacement of the column, replacement of the analytical procedure, change of the detection wavelength, change of the column temperature, and the like were not carried out in the middle, except for cleaning of the column with methanol, and further, operations such as reconstitution of the mobile phase and re-degassing were not carried out.

Example 1 analysis of palmitoyl tripeptide-1

With reference to the above analysis conditions, the palmitoyl tripeptide-1 product of the company was analyzed by HPLC, and the results are shown in FIG. 1: the retention time of palmitoyl tripeptide-1 is 9.585min.

Example 2 analysis of palmitoyl tripeptide-5

As a result of HPLC analysis of palmitoyl tripeptide-5, a product of this company, under the above analysis conditions, the retention time of palmitoyl tripeptide-5 was 8.196min, as shown in FIG. 2.

Example 3 analysis of palmitoyl tripeptide-8

As a result of HPLC analysis of palmitoyl tripeptide-8, a product of this company, under the above analysis conditions, the retention time of palmitoyl tripeptide-8 was 6.478min, as shown in FIG. 3.

Example 4 analysis of palmitoyl tetrapeptide-7

As a result of HPLC analysis of palmitoyl tetrapeptide-7, a product of this company, under the above analysis conditions, the retention time of palmitoyl tetrapeptide-7 was 8.212min, as shown in FIG. 4.

Example 5 analysis of palmitoyl pentapeptide-4

As a result of HPLC analysis of palmitoyl pentapeptide-4, a product of this company, under the above analysis conditions, the retention time of palmitoyl pentapeptide-4 was 9.287min, as shown in FIG. 5.

Comparative example 1

The palmitoyl tripeptide-5 sample was separated and measured for retention time without adjusting pH in the same manner as in example 2, except that methanol was replaced with acetonitrile in the mobile phase, and as a result, as shown in fig. 6, it was revealed that a continuous peak appeared, presumably due to improper selection of the mobile phase, to decrease the separation effect and the calculation accuracy of purity.

Comparative example 2

The retention time of the palmitoyl pentapeptide-4 sample was isolated and measured in the same manner as in example 5, except that the mobile phase methanol was changed to acetonitrile, the pH was not adjusted, and the sample was dissolved with acetonitrile containing 0.1% trifluoroacetic acid, and as a result, as shown in fig. 7, it can be seen that 3 peaks appeared in the peak shape, presumably due to the change in the mobile phase, and the sample may be insufficiently dissolved when the sample was dissolved with acetonitrile containing 0.1% trifluoroacetic acid, thereby decreasing the separation effect and the calculation accuracy of purity.

The result shows that the HPLC analysis method applicable to various palmitoyl peptides can continuously separate and measure the palmitoyl peptide of the embodiment 1~5, the peak shapes of all peptides in the chromatogram are clear, the technology can greatly save the time for preparing the mobile phase and the waiting time consumed during peptide analysis switching, not only can save the time cost, but also can save the space occupied by mobile phase storage and chromatographic bottle storage and the labor consumption in the processes of preparation and switching, and therefore, the method is more beneficial to the realization of full-process automation.

By using the HPLC analysis method applicable to various palmitoyl peptides, the five palmitoyl peptides of the embodiment 1~5 have completely unified analysis method, and each palmitoyl peptide can be well verified.

In conclusion, the HPLC analysis method suitable for various palmitoyl peptides is simple to operate, saves a large amount of time cost and economic cost when various palmitoyl peptides are continuously separated and determined, greatly simplifies the complexity of operation, and has universality on the palmitoyl peptides.

Claims

1. An HPLC separation method suitable for various palmitoyl peptides, which is characterized in that:

the first step is as follows: preparing a plurality of solutions of palmitoyl peptide samples to be detected, wherein the palmitoyl peptide samples are single palmitoyl peptide;

the second step: setting liquid phase chromatographic conditions according to the type of the prepared to-be-detected palmitoyl peptide sample;

the third step: preparing a mobile phase for analysis;

wherein:

for the mobile phase for analysis, the mobile phase A is 0.2% phosphoric acid aqueous solution, and the pH is adjusted to 3.5 by triethylamine; the mobile phase B is methanol;

the analytical procedure was isocratic elution, elution concentration: mobile phase A:15%, mobile phase B:85 percent;

the chromatographic column is a chromatographic column with C18 as a filler;

the palmitoyl peptide is palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl tripeptide-8, palmitoyl tetrapeptide-7, and palmitoyl pentapeptide-4.

2. An HPLC separation method for palmitoyl peptides according to claim 1, characterized in that: the palmitoyl peptide sample to be detected is prepared according to the following proportion, 0.008 to 0.012g of palmitoyl peptide is weighed, 0 to 8.5mL of methanol is added according to needs, 0.2mL of phosphoric acid is added, the palmitoyl peptide is dissolved and then is supplemented to 10mL by adding ultrapure water, and the solution of the palmitoyl peptide sample to be detected with the concentration of 0.0008 to 0.0012g/mL is obtained after dissolution.

3. An HPLC separation method for palmitoyl peptides according to claim 2, characterized in that: the concentration of the palmitoyl peptide sample to be detected is 0.001g/ml.

4. An HPLC separation method for various palmitoyl peptides, according to claim 1 or 2, characterized in that: the wavelength range is 200 to 300nm.

5. An HPLC separation method for various palmitoyl peptides, according to claim 1 or 2, characterized in that: the flow rate was 1mL/min.

6. An HPLC separation method for various palmitoyl peptides, according to claim 1 or 2, characterized in that: the temperature of the chromatographic column is room temperature, the sample injection amount is 8 to 15 mu L, the temperature of the sample injector is ambient temperature, and the collection time of the sample to be detected is 10 to 30min.