CN115267013A - HPLC analysis method suitable for various palmitoyl peptides - Google Patents
HPLC analysis method suitable for various palmitoyl peptides Download PDFInfo
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- G01N30/02—Column chromatography
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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: 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 a chromatographic column for 1 to 5min by adopting 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
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 HPLC analysis methods for peptides have been reported, however, few analysis methods for palmitoyl peptides have been reported. The palmitoyl peptide has a palmitoyl modification more than a normal peptide, so that the properties of the peptide are greatly changed, and the conventional peptide analysis method is not suitable for the palmitoyl peptide. Prior art document CN 106546673A discloses a method for separating palmitoyl pentapeptide-3 by high performance liquid chromatography, however, this method adds trifluoroacetic acid in the mobile phase in order to improve the peak shape of chromatographic peak and separation effect. This results in the end product having acid or trifluoroacetic acid residues which make the product acidic and noticeably tart, 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, there is no more than a single common HPLC method for sequential separation and determination of multiple 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 proportion of acetonitrile or methanol and water is adjusted to achieve the purpose of separation. 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, and are not generally applicable to most of peptides or proteins, and when inorganic salts are used, if the washing is incomplete, the instrument or chromatographic column is easily blocked, which brings trouble 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 and cost waste caused by the procedures of replacing or cleaning a chromatographic column, replacing a mobile phase, re-balancing and the like when a plurality of palmitoyl peptide samples are continuously subjected to separation and measurement by an HPLC method.
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 is characterized in that:
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 determining other undetected palmitoyl peptide samples to be detected;
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 of various palmitoyl peptides, thereby reducing the consumption of manpower and material resources during analysis. When different palmitoyl peptides are analyzed, the chromatographic column is not required to be replaced, only the chromatographic column is required to be cleaned, the analysis program is not required to be replaced, the detection wavelength is not required to be changed, the temperature of the chromatographic column is not required to be changed, the mobile phase is not required to be prepared again, the degassing is not required to be carried out again, and the analysis and the 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 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 a chromatographic column for 1 to 5min by adopting 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 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 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 is selected in consideration of complete dissolution of the peptide and no influence of the selected solvent on analysis of the peptide, namely, light absorption of the solvent and light absorption of the peptide are not overlapped, and the solvent does not corrode an HPLC analysis system. Therefore, the formulation of palmitoyl peptide samples becomes of particular importance 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 the palmitoyl tripeptide 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 affected 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: all palmitoyl peptides were scanned at full wavelength, and most palmitoyl peptides absorbed light strongly at 215nm, so 215nm is most preferred as the detection wavelength for 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 is not to be construed as being limited thereto, in conjunction 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. 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, unless otherwise specified.
Examples
Instruments and conditions:
an Agilent1260InfinityII LC high performance liquid chromatograph and an OpenLabCDS2 software system are adopted; taking agent ZORBAX SB-C18 (250 multiplied by 4.6 mm) as a separation column, and keeping the column temperature at 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 is 25 ℃.
Sample introduction amount: 10uL.
And (3) analysis program: isocratic elution was performed using the elution concentrations described above.
Examples 1 to 5 were continuously subjected to high performance liquid chromatography analysis under the above-mentioned chromatographic conditions, and chromatograms were recorded, and when the test sample was replaced for the test, operations such as replacement of the column, replacement of the analysis program, 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-deaeration 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 was 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 palmitoyl tripeptide-8 showed a retention time of 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, with reference to the above analysis conditions, the retention time of palmitoyl pentapeptide-4 was 9.287min, as shown in FIG. 5.
Comparative example 1
With the exception of changing methanol to acetonitrile in the mobile phase and without adjusting pH, the separation of the palmitoyl tripeptide-5 sample was measured for retention time in the same manner as in example 2, 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 results show that the HPLC analysis method applicable to various palmitoyl peptides can be used for continuously separating and measuring the palmitoyl peptides in examples 1 to 5, the peak shapes of the peptides in a chromatogram are clear, the technology can greatly save the time for preparing a 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.
With the HPLC analysis method applicable to various palmitoyl peptides, the five palmitoyl peptides of examples 1 to 5 were completely unified in analysis method, and each palmitoyl peptide was 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 (7)
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, 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: a mobile phase A:15%, mobile phase B:85 percent.
2. An HPLC analysis method for various 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 analysis method for various palmitoyl peptides, according to claim 2, characterized in that: the concentration of the palmitoyl peptide sample to be detected is 0.001g/ml.
4. The HPLC analytical method for various palmitoyl peptides of claims 1 or 2, characterized in that: the wavelength range is 200 to 300nm.
5. An HPLC analysis method for various palmitoyl peptides, according to claim 1 or 2, characterized in that: the chromatographic column is a C18 packing chromatographic column, and the flow rate is 1mL/min.
6. An HPLC analysis 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.
7. The HPLC analytical method for various palmitoyl peptides of claims 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.
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| CN116381091A (en) * | 2023-04-07 | 2023-07-04 | 广州美域医学检验有限公司 | Method for detecting palmitoyl tripeptide-5 content |
| CN117740993A (en) * | 2023-12-18 | 2024-03-22 | 广州美域医学检验有限公司 | Method for detecting peptide substances in cosmetics and application thereof |
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| RALUCA-IOANA CHIRITA ET AL: "Development of a LC–MS/MS method to monitor palmitoyl peptides content in anti-wrinkle cosmetics", 《ANALYTICA CHIMICA ACTA》 * |
| 王小青等: "棕榈酰五肽-3的固相合成", 《承德医学院学报》 * |
| 陈意光等: "液相色谱-串联质谱法测定护肤类化妆品中5种棕榈酰多肽", 《理化检验(化学分册)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116381091A (en) * | 2023-04-07 | 2023-07-04 | 广州美域医学检验有限公司 | Method for detecting palmitoyl tripeptide-5 content |
| CN117740993A (en) * | 2023-12-18 | 2024-03-22 | 广州美域医学检验有限公司 | Method for detecting peptide substances in cosmetics and application thereof |
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| CN115267013B (en) | 2022-12-13 |
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