CN115453005A - HPLC analysis method suitable for continuously analyzing various palmitoyl peptides - Google Patents
HPLC analysis method suitable for continuously analyzing various palmitoyl peptides Download PDFInfo
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Abstract
The invention relates to an HPLC analysis method suitable for continuously analyzing various palmitoyl peptides, which is characterized by comprising the following steps of: 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 continuously analyzing a plurality of palmitoyl peptides can continuously analyze a plurality of palmitoyl peptides and continuously perform concentration determination on a plurality of palmitoyl peptide samples with unknown concentrations.
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 continuously analyzing 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 CN106546673A 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 sour, while some peptides are actually unstable under acidic conditions, affecting its shelf life. Furthermore, trifluoroacetic acid is a strong acid, which has a high requirement on the tolerance of chromatographic columns, and a reinforced chromatographic column is required because the general chromatographic column cannot bear the strong acid.
In addition, it is also rare in the prior art to use a common HPLC method for continuous analytical determination of a variety of peptides. Generally, each peptide corresponds to a specific method, so that the preparation work of the mobile phase is complicated, the required reagents are 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 and 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 within 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, and are not generally applicable to most peptides or proteins. In the past work, the situation that several peptides cannot be completely separated easily occurs when the peptides are analyzed, and the complete separation is required when the peptide content is analyzed, otherwise, the peptide content 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 present inventors have conducted extensive studies for a long time and found an HPLC analysis method which can continuously analyze a plurality of palmitoyl peptides using a phosphoric acid solution adjusted to a specific pH with triethylamine as a specific mobile phase. However, although this method allows for the continuous analysis of a variety of palmitoyl peptides, several drawbacks remain. For example, when palmitoyl tripeptide-1 or the like is continuously measured by this method, the theoretical plate number ratio is low, and a linear pattern can be obtained, but the error is large. The theoretical plate number is one of column efficiency parameters of the chromatography and is used for quantitatively expressing the separation efficiency of the chromatographic column. When the lengths of the chromatographic columns are the same, the larger the number of theoretical plates is, the higher the column efficiency is, the better the peak shape is, and accordingly, a more accurate linear graph can be obtained, so that a more accurate standard curve is provided for the content determination of palmitoyl peptide in an unknown sample.
Therefore, a more general high performance liquid chromatography is urgently needed to be researched and searched, which not only has universality for more kinds of palmitoyl peptides, but also can provide better peak shape to obtain higher theoretical plate number, so that a more accurate standard curve can be provided for measuring the content of palmitoyl peptide in a sample with unknown concentration, and further the continuous measurement of the content of palmitoyl peptide in a plurality of samples with unknown concentration can be realized.
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 continuously carrying out HPLC method analysis and measurement on a plurality of palmitoyl peptide samples.
It is a further object of the present invention to optimize the analytical determination process for a plurality of palmitoyl peptide samples by continuous HPLC method, which provides a better peak profile to obtain a higher number of theoretical plates, thereby providing a more accurate standard curve for determining the content of palmitoyl peptide in unknown samples, so that the content of palmitoyl peptide in a plurality of samples of unknown concentration can be continuously measured.
In addition, the invention solves the problem that trifluoroacetic acid added for improving the peak shape of chromatographic peak in the prior art burdens the chromatographic column.
Means for solving the problems
The present invention relates to:
1. an HPLC analysis method suitable for continuous analysis of a plurality of palmitoyl peptides, 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: setting liquid phase chromatographic conditions according to the type of the prepared palmitoyl peptide sample to be detected;
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 by adopting a mobile phase B for 1-5 min;
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 20mM KH 2 PO 4 An aqueous solution; the mobile phase B is methanol;
the analytical procedure was isocratic elution, elution concentration: mobile phase A:20%, mobile phase B:80 percent.
2. The HPLC analysis method according to item 1, which is suitable for continuous analysis of various palmitoyl peptides, characterized by: the palmitoyl peptide sample to be detected is prepared according to the following proportion, 0.001-0.030 g of palmitoyl peptide is weighed, 0-8.5 mL of methanol is added according to the requirement, 0.2mL of phosphoric acid is added, ultrapure water is added to supplement to 10mL after the palmitoyl peptide is dissolved, and the solution of the palmitoyl peptide sample to be detected with the concentration of 0.0001-0.0030 g/mL is obtained after the palmitoyl peptide is dissolved.
3. The HPLC analysis method according to item 1 or 2, which is suitable for continuously analyzing a plurality of palmitoyl peptides, characterized in that: the wavelength range is 200-300 nm.
4. The HPLC analysis method according to item 1 or 2, which is suitable for continuously analyzing a plurality of palmitoyl peptides, characterized in that: the chromatographic column is a C18-packed chromatographic column with the flow rate of 1mL/min.
5. The HPLC analysis method suitable for continuously analyzing a plurality of 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 8-15 mu L, the temperature of the sample injector is ambient temperature, and the collection time of the sample to be detected is 10-30 min.
6. The HPLC analysis method suitable for continuously analyzing a plurality of 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 an HPLC analysis method suitable for continuously analyzing various palmitoyl peptides, which unifies the analysis methods of various palmitoyl peptides and reduces the consumption of manpower and material resources during analysis. When different palmitoyl peptides are analyzed, the chromatographic column does not need to be replaced, only the chromatographic column needs to be cleaned, the detection wavelength does not need to be changed, the temperature of the chromatographic column does not need to be changed, and the analysis and the purity calculation of various palmitoyl peptides can be realized without preparing a mobile phase again and degassing again.
Secondly, the HPLC analysis method suitable for continuously analyzing various palmitoyl peptides improves the peak shape while realizing continuous analysis and determination of various palmitoyl peptides, greatly improves the number of theoretical plates when the length of a chromatographic column is the same under the condition of not adding trifluoroacetic acid, thereby obtaining a more accurate linear graph, providing a more accurate standard curve for content determination of palmitoyl peptides in unknown samples, and further realizing continuous calibration of various palmitoyl peptides with unknown concentration.
The HPLC analysis method suitable for continuously analyzing various palmitoyl peptides is simple to operate, saves a large amount of time cost and economic cost when continuously analyzing and determining various 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 peptide switching 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 preparation and switching processes, 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 a plot of palmitoyl tripeptide-1 from example 1.
FIG. 3 is an HPLC chromatogram of palmitoyl tripeptide-5 from example 2.
FIG. 4 is a plot of palmitoyl tripeptide-5 from example 2.
FIG. 5 is an HPLC chromatogram of palmitoyl tripeptide-8 from example 3.
FIG. 6 is a plot of palmitoyl tripeptide-8 from example 3.
FIG. 7 is an HPLC chromatogram of palmitoyl tetrapeptide-7 in example 4.
FIG. 8 is a plot of palmitoyl tetrapeptide-7 from example 4.
FIG. 9 is an HPLC chromatogram of palmitoyl pentapeptide-4 of example 5.
FIG. 10 is a plot of palmitoyl pentapeptide-4 from example 5.
FIG. 11 is an HPLC chromatogram of palmitoyl tripeptide-1 of comparative example 1.
FIG. 12 is an HPLC chromatogram of palmitoyl tripeptide-5 of comparative example 2.
FIG. 13 is an HPLC chromatogram of palmitoyl pentapeptide-4 of comparative example 3.
Detailed Description
The HPLC analysis method suitable for continuously analyzing 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: setting liquid phase chromatographic conditions according to the type of the prepared palmitoyl peptide sample to be detected;
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 by adopting a mobile phase B for 1-5 min;
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 as follows: phase A:20mM KH 2 PO 4 An aqueous solution; phase B: methanol. The analytical procedure was isocratic elution, elution concentration: mobile phase A:20%, mobile phase B:80 percent. The range is selected to obtain the best analysis results. Although the analysis effect can be achieved by the mobile phase in other ranges, the analysis effect and the analysis efficiency are inferior to those achieved by the mobile phase.
The concentration of the palmitoyl peptide sample to be detected is 0.0001-0.0030 g/ml. Compared with the prior art, the concentration range of continuous measurement is greatly improved, in the measurement range, in order to enable the peak of the target peptide to be completely displayed, the peptide concentration is not suitable to be too high, otherwise the target peak is capped, and the judgment cannot be carried out even if a mixed peak appears at the peak top; 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. Therefore, the concentration of the palmitoyl peptide sample to be detected is 0.0001-0.0030 g/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) than the common peptide, so that the property of the peptide per se is greatly changed, namely the change from water solubility to fat solubility is realized, but few peptides still show water solubility even with the palmitoyl group because the hydrophilicity of the amino acid per se is stronger than the hydrophobicity of the palmitoyl group, such as palmitoyl tripeptide-5. HPLC (high performance liquid chromatography) measurement requires complete dissolution of palmitoyl peptide for detection, but most of palmitoyl peptide cannot be dissolved in water, and the solvent selection needs to consider that the peptide can be completely dissolved and the selected solvent does not influence the analysis of the peptide, namely the light absorption of the solvent and the light absorption of the peptide do not overlap, 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 0.001-0.030 g of palmitoyl peptide, adding 0-8.5 mL of methanol according to needs, adding 0.2mL of phosphoric acid, dissolving the palmitoyl peptide, and adding ultrapure water to supplement to 10mL to obtain 0.0001-0.0030 g/mL of solution of the palmitoyl peptide sample to be detected. Particularly, 0.001-0.030 g of palmitoyl tripeptide-5 is weighed and dissolved in 10mL of ultrapure water to obtain a solution of the palmitoyl tripeptide-5 sample to be detected with the concentration of 0.0001-0.0030 g/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 Agilent ZORBAX SB-C18.
Isocratic elution is adopted, and the elution concentration is as follows: 20% of phase A; 80% 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 10-30 min, 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. 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 Agilent 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:
five groups of samples were prepared by weighing palmitoyl tripeptide-1 (example 1), palmitoyl tripeptide-8 (example 3) and palmitoyl tetrapeptide-7 (example 4) in amounts of 0.002g, 0.004g, 0.006g, 0.008g and 0.01g, and palmitoyl tripeptide-5 (example 2, comparative example 1) and palmitoyl pentapeptide-4 (example 5, comparative example 2) in amounts of 0.005g, 0.010g, 0.015g, 0.020g and 0.030g, respectively. Each sample of each group of the weighed palmitoyl tripeptide-1 (example 1), palmitoyl tripeptide-8 (example 3), palmitoyl tetrapeptide-7 (example 4) and palmitoyl pentapeptide-4 (example 5) was added with 8.5mL of methanol and 0.2mL of phosphoric acid, and dissolved and then supplemented with ultrapure water to 10mL to obtain peptide solutions of 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L and 1.0g/L or peptide solutions of 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L and 3.0g/L for each group of samples. Respectively dissolving 0.005g, 0.010g, 0.015g, 0.020g and 0.030g of palmitoyl tripeptide-5 in 10mL of ultrapure water to obtain 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L and 3.0g/L peptide solutions of palmitoyl tripeptide-5. The five peptides are all products from Proteo Biotech (Guangzhou) Limited, which are verified by Beijing Baishipekko Paeko Biotech, inc. for peptide sequence and molecular weight.
Mobile phase: a:20mM KH 2 PO 4 An aqueous solution; b: methanol, HPLC grade.
Elution concentration: a mobile phase A:20%, mobile phase B:80 percent.
Flow rate: 1.0mL/min.
Temperature: at 25 ℃.
Sample introduction amount: 10uL.
And (3) analysis program: isocratic elution; elution concentration: mobile phase A:20%, mobile phase B:80 percent.
The high performance liquid chromatography analyses of examples 1 to 5 were continuously performed under the above-mentioned chromatographic conditions, and chromatograms were recorded, and when the test samples were replaced and tested, operations such as replacement of the column, change of the detection wavelength, change of the column temperature, and the like were not performed in the middle, except that the column was washed with methanol, and operations such as reconstitution of the mobile phase and re-deaeration were not performed.
Example 1 analysis of palmitoyl tripeptide-1
With reference to the above analysis conditions, HPLC analysis was performed on peptide solutions of palmitoyl tripeptide-1, a product of this company, at concentrations of 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L, and 1.0g/L, respectively, and the results are shown in FIG. 1 and in FIG. 2, as linear plots. The retention time and the number of theoretical plates of palmitoyl tripeptide-1 at each concentration are shown in the following table.
| Concentration g/L | Retention time min | Number of theoretical plates per unit |
| 0.2 | 2.592 | 9699.77 |
| 0.4 | 2.592 | 9081.18 |
| 0.6 | 2.580 | 8605.70 |
| 0.8 | 2.579 | 8141.41 |
| 1.0 | 2.606 | 7727.90 |
Example 2 analysis of palmitoyl tripeptide-5
With reference to the above analysis conditions, HPLC analysis was performed on peptide solutions of palmitoyl tripeptide-5, a product of this company, at concentrations of 0.5g/L, 1g/L, 1.5g/L, 2g/L, and 3g/L, respectively, and the results are shown in FIG. 3 and FIG. 4, which are linear plots. The retention time and the number of theoretical plates of palmitoyl tripeptide-5 at each concentration are shown in the following table.
| Concentration g/L | Retention time min | Number of theoretical plates per unit |
| 0.5 | 14.894 | 4308.39 |
| 1.0 | 14.835 | 3844.97 |
| 1.5 | 14.719 | 3324.51 |
| 2.0 | 14.597 | 3346.70 |
| 3.0 | 14.241 | 3305.75 |
Example 3 analysis of palmitoyl tripeptide-8
HPLC analysis of peptide solutions containing the palmitoyl tripeptide-8, a product of this company, at concentrations of 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L and 1.0g/L, was performed under the above analytical conditions, and the results are shown in FIG. 5 and in FIG. 6 as linear plots. The retention time and the number of theoretical plates of the palmitoyl tripeptide-8 at each concentration are shown in the table below.
| Concentration g/L | Retention time min | Number of theoretical plates per unit |
| 0.2 | 11.890 | 8702.97 |
| 0.4 | 11.790 | 7128.74 |
| 0.6 | 11.723 | 6084.31 |
| 0.8 | 11.656 | 5275.24 |
| 1.0 | 11.605 | 4771.69 |
Example 4 analysis of palmitoyl tetrapeptide-7
HPLC analysis was performed on peptide solutions of palmitoyl tetrapeptide-7, a product of this company, at concentrations of 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L, and 1.0g/L, respectively, under the above analysis conditions, and the results are shown in FIG. 7 and in FIG. 8, which are linear plots. The retention time and the number of theoretical plates of palmitoyl tetrapeptide-7 at each concentration are shown in the following table.
| Concentration g/L | Retention time min | Number of theoretical plates per unit |
| 0.2 | 13.934 | 10633.37 |
| 0.4 | 13.963 | 8519.72 |
| 0.6 | 13.952 | 10417.15 |
| 0.8 | 13.956 | 10259.01 |
| 1.0 | 13.967 | 10094.72 |
Example 5 analysis of palmitoyl pentapeptide-4
With reference to the above analysis conditions, HPLC analysis was performed on peptide solutions having concentrations of palmitoyl pentapeptide-4, a product of this company, of 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L, and 3.0g/L, respectively, and the results are shown in FIG. 9 and a linear plot in FIG. 10. The retention time and the number of theoretical plates of palmitoyl pentapeptide-4 at each concentration are shown in the following table.
| Concentration g/L | Retention time min | Number of theoretical plates per unit |
| 0.5 | 11.119 | 6991.48 |
| 1.0 | 11.069 | 6234.31 |
| 1.5 | 10.985 | 5140.46 |
| 2.0 | 10.909 | 4459.21 |
| 3.0 | 10.772 | 3587.07 |
Comparative example 1
Except that the mobile phase a:20mM KH 2 PO 4 HPLC analysis of a 1.0g/L peptide solution of a sample of palmitoyl tripeptide-1 was performed in the same manner as in example 1, except that the aqueous solution was replaced with a 0.2% aqueous phosphoric acid solution adjusted to pH 3.5 with triethylamine, and the results are shown in FIG. 11, with a retention time and a theoretical plate number of 9.585min and 941.18, respectively.
Comparative example 2
Except that the mobile phase a:20mM KH 2 PO 4 HPLC analysis of a 1.0g/L peptide solution of a palmitoyl tripeptide-5 sample was performed in the same manner as in example 2, except that the aqueous solution was replaced with a 0.2% aqueous phosphoric acid solution adjusted to pH 3.5 with triethylamine, and the results are shown in FIG. 12.
Comparative example 3
Except that the mobile phase a:20mM KH 2 PO 4 HPLC analysis of a 1.0g/L peptide solution of a palmitoyl pentapeptide-4 sample was performed in the same manner as in example 5 except that the aqueous solution was replaced with a 0.2% aqueous phosphoric acid solution adjusted to pH 3.5 with triethylamine, and the retention time and the theoretical plate number were 9.287min and 1109.23, respectively, as shown in FIG. 13.
As can be seen from the results of comparing example 1 with comparative example 1, example 2 with comparative example 2, and example 5 with comparative example 3, the number of theoretical plates was greatly increased, the peak shape was greatly improved, the concentration range of application was significantly increased, and good linearity was obtained when analytical measurements were performed by the HPLC method of the present invention.
The results show that: the HPLC analysis method suitable for continuously analyzing a plurality of palmitoyl peptides can continuously analyze a plurality of palmitoyl peptides, and can also continuously measure the concentration of a plurality of palmitoyl peptide samples with unknown concentration based on the standard curve of the method.
The technology of the invention 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 consumption of labor force in the process of preparation and switching, thereby leading the method to be more beneficial to the realization of whole-process automation.
With the HPLC analysis method of the present invention, which is suitable for continuous analysis of various palmitoyl peptides, the five palmitoyl peptides of examples 1 to 5 are completely unified in analysis method, each palmitoyl peptide can be well verified, and a large number of samples can be continuously measured, thereby satisfying industrial batch measurement.
In conclusion, the HPLC analysis method suitable for continuously analyzing various palmitoyl peptides is simple to operate, saves a large amount of time cost and economic cost when continuously analyzing and determining various palmitoyl peptides, greatly simplifies the complexity of operation, and has universality on palmitoyl peptides.
Claims (6)
1. An HPLC analysis method suitable for continuously analyzing a plurality of palmitoyl peptides, 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 palmitoyl peptide sample to be detected;
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 20mM KH 2 PO 4 An aqueous solution; the mobile phase B is methanol;
the analytical procedure was isocratic elution, elution concentration: mobile phase A:20%, mobile phase B:80 percent.
2. The HPLC analytical method suitable for the sequential analysis of a plurality of palmitoyl peptides of claim 1, characterized in that: the palmitoyl peptide sample to be detected is prepared according to the following proportion, 0.001 to 0.030g 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 0.0001 to 0.0030g/mL is obtained after dissolution.
3. The HPLC analytical method suitable for the sequential analysis of a plurality of palmitoyl peptides of claims 1 or 2, characterized in that: the wavelength range is 200 to 300nm.
4. The HPLC analytical method suitable for the sequential analysis of a plurality of palmitoyl peptides of claims 1 or 2, characterized in that: a C18-packed column was used at a flow rate of 1mL/min.
5. The HPLC analytical method suitable for the sequential analysis of a plurality of palmitoyl peptides of claims 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.
6. An HPLC analysis method suitable for the continuous analysis of various palmitoyl peptides, according to claim 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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| CN106546673A (en) * | 2016-10-20 | 2017-03-29 | 吉尔生化（上海）有限公司 | A kind of method that utilization high performance liquid chromatography separates palmityl Wushengtai 3 |
| CN110286202A (en) * | 2019-05-25 | 2019-09-27 | 江苏食品药品职业技术学院 | A kind of quality control detector for polypeptide drugs |
| WO2020245800A1 (en) * | 2019-06-07 | 2020-12-10 | Waters Technologies Corporation | Improving rplc-based peptide mapping chromatographic performance using metal chelators as mobile phase additives |
| CN113912871A (en) * | 2021-12-13 | 2022-01-11 | 浙江湃肽生物有限公司深圳分公司 | Separation method of palmitoyl pentapeptide-4 |
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| CN106546673A (en) * | 2016-10-20 | 2017-03-29 | 吉尔生化（上海）有限公司 | A kind of method that utilization high performance liquid chromatography separates palmityl Wushengtai 3 |
| CN110286202A (en) * | 2019-05-25 | 2019-09-27 | 江苏食品药品职业技术学院 | A kind of quality control detector for polypeptide drugs |
| WO2020245800A1 (en) * | 2019-06-07 | 2020-12-10 | Waters Technologies Corporation | Improving rplc-based peptide mapping chromatographic performance using metal chelators as mobile phase additives |
| CN113912871A (en) * | 2021-12-13 | 2022-01-11 | 浙江湃肽生物有限公司深圳分公司 | Separation method of palmitoyl pentapeptide-4 |
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