High performance liquid chromatography detection method of antibacterial peptide A24

Abstract

The invention discloses a high performance liquid chromatography detection method of an antibacterial peptide A24. According to the invention, a high performance liquid chromatography detection method is established aiming at the fungal defensin peptide A24, and concentration determination of the antibacterial peptide in different tissues can be realized. The extraction and purification method disclosed by the invention is high in extraction rate and recovery rate and good in repeatability, so that the feasibility, stability and accuracy of HPLC detection are improved, and a foundation is laid for research on pharmacokinetics of the fungal defensin peptide and the derived peptide A24 of the fungal defensin peptide.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptide detection and analysis, in particular to a high performance liquid chromatography detection method for antimicrobial peptide A24. Background Art

[0002] Compared with traditional antibiotics, antimicrobial peptides (AMPs) have the advantages of fast bactericidal speed and low resistance to drug resistance. As antibiotic candidates, they have attracted extensive attention from researchers (ZASLOFF et al., 1987). Currently, thousands of AMPs are included in the AMP database, and most AMPs have been patented. They have great potential for candidate drug development. However, only a few AMPs are currently used as drugs. The underlying reasons are thought-provoking (ALEXANDRE et al., 2014). Antimicrobial peptides are unstable in vivo and are easily degraded by a variety of matrices, enzymes, factors, etc. They are not easy to cause drug residues and drug resistance. The disadvantage is that they degrade quickly in vivo, which brings great difficulties to detection, especially in a variety of biological matrices. It is difficult to be detected, which causes great difficulties for drug characterization and metabolic research in vivo (KANTHAWONG et al., 2010). At present, the main methods used for peptide detection are serological detection methods such as ELISA, mass spectrometry detection methods, and microbial detection methods. LC–MS / MS methods have been established for the detection of plectasin, defensin, LR8P and liraglutide in plasma (ZHENG et al., 2022; SCHNEIDER et al., 2010). Currently, there are no reports on the detection of fungal defensin peptide A24. Summary of the invention

[0003] The purpose of the present invention is to provide a high performance liquid chromatography detection method for antimicrobial peptide A24.

[0004] In order to achieve the purpose of the present invention, the present invention provides a high performance liquid chromatography (HPLC) detection method of antimicrobial peptide A24, which uses high performance liquid chromatography to detect antimicrobial peptide A24 in liquid samples or tissue samples (the amino acid sequence of antimicrobial peptide A24 can be found in CN115969956A).

[0005] Considering that the antimicrobial peptide A24 may be administered intravenously and into the uterus in the future, the liquid sample includes aqueous solution and plasma; the tissue sample includes but is not limited to uterine tissue.

[0006] The method comprises the following steps: (1) The sample is pre-treated to obtain the sample to be tested; (2) Prepare standard sample solutions of antimicrobial peptide A24 of different concentrations, perform high performance liquid chromatography detection, and then draw a standard curve with the concentration of antimicrobial peptide A24 as the horizontal axis and the peak area as the vertical axis; (3) The sample to be tested is subjected to high performance liquid chromatography detection under the same conditions as step (2), and the content of antimicrobial peptide A24 in the sample to be tested is obtained according to the standard curve.

[0007] Furthermore, the chromatograph used for high performance liquid chromatography detection is Agilent 1260HPLC, UV detector, chromatographic column is ZORBAX Eclipsc Plus C18, specification 4.6 mm × 250 mm, 5 μm, acidic organic solvent is used as mobile phase A, acidic aqueous solution is used as mobile phase B, gradient elution is performed; column temperature is 25-40°C; detection wavelength is 220-280 nm; loading speed is 0.5-1.5 mL / min. Preferably, the column temperature is 30°C; the detection wavelength is 278 nm; the loading speed is 0.8 mL / min; and the injection volume is 10 μL.

[0008] Furthermore, the acidic aqueous solution is selected from at least one of trifluoroacetic acid, acetic acid, phosphoric acid, hydrochloric acid, formic acid aqueous solution, etc., preferably 0.01% to 1% trifluoroacetic acid aqueous solution, more preferably 0.1% trifluoroacetic acid aqueous solution.

[0009] Furthermore, the organic solvent is selected from at least one of acetonitrile, methanol, ethanol, isopropanol, etc., preferably acetonitrile.

[0010] More preferably, the acidic organic solvent is acetonitrile containing 0.1% trifluoroacetic acid.

[0011] Furthermore, the elution gradient of the mobile phase A is 10%-40%, and the elution gradient of the mobile phase B is 90%-60%.

[0012] The gradient elution conditions were as follows:

[0013] Wherein, the phase A is acetonitrile containing 0.1% trifluoroacetic acid, and the phase B is 0.1% trifluoroacetic acid aqueous solution.

[0014] Furthermore, the pretreatment method of the plasma sample in step (1) includes: adding 1% trifluoroacetic acid aqueous solution to the plasma, filtering through a 0.22 µm filter membrane, and using the sample as a test sample.

[0015] Furthermore, the pretreatment method of the uterine tissue sample in step (1) includes: adding 3 mL of 0.1 M PBS buffer to 0.1 g of uterine tissue sample, homogenizing and mixing, adding 3-5 mL of extracting solution, vortexing and mixing, and centrifuging to collect the supernatant; adding an equal volume of n-hexane to the supernatant, vortexing and mixing, and centrifuging to collect the lower layer of liquid; then purifying the sample with a purification column, concentrating the purified sample, and re-dissolving it with ultrapure water as a sample to be tested.

[0016] The extract is prepared by mixing 1L of 0.1% formic acid aqueous solution with 400mL of methanol.

[0017] Furthermore, the purification column is selected from a WCX purification column, a MAX purification column, an MHA purification column or an HLB purification column, etc., preferably an HLB purification column.

[0018] Further, the purification and concentration method includes: 1) Activate the HLB purification column with methanol, ultrapure water or ammoniated methanol, preferably methanol; 2) Use deionized water or McFarland buffer for balance, preferably McFarland buffer; 3) eluting with a mixed solvent of acetonitrile and water, wherein the content of acetonitrile is 25% to 100%, preferably 40%; 4) The obtained eluate is concentrated and then reconstituted with ultrapure water to serve as the sample to be tested; The concentration was carried out using a constant temperature water bath nitrogen blowing apparatus with a nitrogen blowing temperature of 40±5°C and a time of 30 to 50 minutes.

[0019] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) The present invention establishes a high performance liquid chromatography detection method for fungal defensin peptide A24, laying a foundation for the study of the pharmacokinetics of fungal defensin peptide and its derivative peptide A24.

[0020] (ii) Sample pretreatment is a prerequisite for HPLC detection. This method can measure the concentration of antimicrobial peptides in different tissues.

[0021] (iii) The extraction and purification method of the present invention has high extraction rate and recovery rate and good repeatability, thereby improving the feasibility, stability and accuracy of HPLC detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is an investigation of the specificity of high performance liquid chromatography detection in a preferred embodiment of the present invention.

[0023] Figure 2 This is a standard curve established based on the peak area and the concentration of antimicrobial peptide A24 in a preferred embodiment of the present invention.

[0024] Figure 3 1 is a HPLC chromatogram of blank uterine matrix and added antimicrobial peptide A24 in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0025] In view of the shortcomings of existing antimicrobial peptide drug detection methods, the present invention provides a fungal defensin peptide high performance liquid chromatography detection method, which can be used to detect antimicrobial peptide A24 in aqueous solution, plasma or uterine tissue, and pre-treat the sample to meet the requirements of high performance liquid chromatography detection.

[0026] The present invention adopts the following technical solution: The invention provides a fungal defensin peptide high performance liquid chromatography detection method, the adopted chromatograph is Agilent 1260 HPLC, the ultraviolet detector is a ZORBAX Eclipsc Plus C18 (4.6 mm×250 mm, 5 μm) chromatographic column, an acidic organic solvent is used as mobile phase A, an acidic aqueous solution is used as mobile phase B, and gradient elution is performed.

[0027] Furthermore, the acidic aqueous solution is selected from at least one of trifluoroacetic acid, acetic acid, phosphoric acid, hydrochloric acid and formic acid, preferably trifluoroacetic acid, with a content ranging from 0.01% to 1%, preferably 0.1%.

[0028] Furthermore, the organic solvent is at least one of acetonitrile, methanol, ethanol and isopropanol, preferably acetonitrile.

[0029] Furthermore, the elution gradient of the mobile phase A is 10%-40%, and the elution gradient of the mobile phase B is 90%-60%.

[0030] Furthermore, during the gradient elution process, the column temperature is 25-40°C, preferably 30°C; at the same time, the detection wavelength is 220-280 nm, preferably 278 nm; the loading speed is 0.5-1.5 mL / min, preferably 0.8 mL / min.

[0031] Furthermore, the detection of antimicrobial peptide A24 in plasma and uterus requires that the plasma and uterine tissue be pre-treated to meet the requirements of high performance liquid chromatography detection, which is mainly divided into two parts: 1) The plasma sample pretreatment method is as follows: 1% trifluoroacetic acid is added to the plasma, and the plasma is filtered through a 0.22 µm filter membrane to obtain a sample for high performance liquid chromatography detection.

[0032] 2) The uterine sample pretreatment method: add 3 mL of 0.1 M PBS buffer to 0.1 g of uterine tissue sample, mix the slurry evenly, add 3-5 mL of extraction solution (1L of 0.1% formic acid aqueous solution and 400 mL of methanol are thoroughly mixed), vortex mix, centrifuge to collect the supernatant, add an equal volume of n-hexane, vortex mix, and centrifuge to collect the lower layer of liquid; then perform sample purification, mainly including: solid phase extraction column activation, equilibration and elution.

[0033] Furthermore, the purification columns are WCX purification columns, MAX purification columns, MHA purification columns and HLB purification columns, preferably HLB purification columns.

[0034] Furthermore, the present invention uses methanol, ultrapure water or ammoniated methanol to activate the HLB purification column, preferably methanol.

[0035] Furthermore, deionized water or McFarland buffer is used for balancing, preferably McFarland buffer.

[0036] Furthermore, a mixed solvent of acetonitrile and water is used for elution, wherein the content of acetonitrile is 25% to 100%, preferably 40%.

[0037] Furthermore, after the sample pretreatment, the antimicrobial peptide A24 eluate is concentrated, and then ultrapure water is added to re-dissolve to obtain an antimicrobial peptide A24 solution; the concentration adopts a constant temperature water bath nitrogen blowing instrument, the nitrogen blowing temperature is 40±5°C, and the time is 30 to 50 minutes; high performance liquid chromatography detects that the A24 target peak has no overlap with impurities, and the purity of the antimicrobial peptide A24 solution is ≥90%.

[0038] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0039] The solid phase extraction column HLB used in the following examples was purchased from Waters Corporation of the United States.

[0040] The preparation method of McFarland buffer is as follows: add 1.625 L of deionized water to 43.143 g of disodium hydrogen phosphate dodecahydrate and 21 g of citric acid, adjust the pH to 4, and then add 60.05 g of disodium ethylenediaminetetraacetate and mix well.

[0041] Example 1 Determination of HPLC Conditions

[0042] First, the HPLC column temperature, sample volume, injection speed, detection wavelength and elution gradient were explored. The temperature was set to 25℃, 30℃ and 35℃ respectively, the sample volume was 10 µL, 20 µL and 40 µL respectively, the injection speed was 0.6 mL / min, 0.8 mL / min, 1 mL / min, 1.2 mL / min, and the detection wavelength was 220-280 nm. The final conditions were determined as follows: chromatographic column: C18; column temperature 30℃; detection wavelength 278 nm; injection volume 10 µL; flow rate 0.8 mL / min; mobile phase gradient elution as shown in Table 1: Table 1 Mobile phase gradient elution

[0043] The results are as follows Figure 1 As shown in the figure, the antimicrobial peptide A24 showed a single target peak at around 11 min, and no mixed peaks appeared in the plasma and uterine samples after pretreatment at around 11 min. The method has strong specificity and can be used for A24 determination (Note: the liquid phase detection results can clearly distinguish the target peak A24, and the peak value is the default output of the instrument and does not affect the observation of the results).

[0044] Example 2 Establishment of standard curve

[0045] Different concentrations of antimicrobial peptide A24 standard aqueous solutions were prepared: 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 µg / mL, with 3 replicates for each concentration. The samples were tested by HPLC as described in Example 1, and the peak area of ​​each concentration was recorded. A standard curve was established with A24 concentration as the abscissa and peak area as the ordinate, and the regression equation and correlation coefficient were obtained. The results are shown in Figure 2 As shown in the figure, A24 showed a good linear relationship at the average values ​​of the diameters of the inhibition zones at the concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 µg / mL, and the linear equation was Y=1.5853x-0.037, R 2 =0.999, showing a good linear relationship, indicating that A24 showed a good linear relationship at 0.25-128 µg / mL.

[0046] Example 3 Precision Testing

[0047] Deionized water was used to prepare the antimicrobial peptide A24 into concentrations of 128, 64, 32, and 16 µg / mL. Six replicates were set within each batch for each concentration, and three parallel replicates were set between batches. The peak areas of each peak were determined according to the HPLC method in Example 1, and the sample concentrations were obtained by substituting the formula into the formula. The RSD was calculated according to the formula: RSD (%) = ((((x1-x 平均值 )+(x2-x 平均值 )+……(x n -x 平均值 )) / (n-1)) / x 平均值 ) × 100

[0048] Where: x represents the measured A24 drug concentration in the sample; It represents the average value of A24 drug concentration measured repeatedly at the same concentration; n represents the number of repeated measurements of the sample.

[0049] The results are shown in Table 2: Table 2 Precision analysis

[0050] Note: Intra-plate variation refers to the difference between replicates of the same concentration in the same plate; inter-plate variation refers to the difference between replicates of the same concentration in different plates.

[0051] Example 4 Recovery rate detection

[0052] The antimicrobial peptide A24 was prepared with deionized water to concentrations of 128, 64, 32, and 16 μg / mL, and 6 parallel replicates were set for each concentration. The peak areas were determined according to the liquid chromatography method in Example 1, and the sample concentration was obtained by substituting it into the formula, which was recorded as the measured drug concentration. At the same time, the actual concentration of the sample was the added drug concentration, and the recovery rate was calculated as follows: Recovery rate (%) = (measured drug concentration / added drug concentration) × 100. The results are shown in Table 3. The recovery rates of A24 with a concentration of 1-128 μg / mL were 87%-102%, and the average recovery rate was 97%, which was close to 1, indicating that the recovery rate was good.

[0053] Table 3 Recovery analysis

[0054] Example 5 Minimum detection limit and quantification limit

[0055] The antimicrobial peptide A24 was prepared with deionized water to concentrations of 32, 16, 8, 4, 2, 1, 0.5, and 0.25 µg / mL, with three replicates for each concentration. The determination was carried out according to the chromatographic conditions in Example 1, with the signal-to-noise ratio (S / N) ≥3 as the detection limit and (S / N) ≥10 as the quantification limit, and the corresponding concentrations were determined. The liquid phase detection results showed that the quantification limit of this method was 2 µg / mL and the detection limit was 1 µg / mL (Note: detection limit: signal-to-noise ratio ≧3; quantification limit: signal-to-noise ratio ≧10). The results showed that the quantification limit of this method was 2µg / mL and the detection limit was 1 µg / mL (Note: detection limit: signal-to-noise ratio ≧3; quantification limit: signal-to-noise ratio ≧10)

[0056] Example 6 Uterine tissue pretreatment

[0057] After preliminary exploration and optimization, mainly including screening of solid phase extraction columns (A24 recovery rate HLB>MAX) and equilibration liquid screening (A24 recovery rate McFarland buffer>extraction liquid), it was determined that the recovery rate after uterine pretreatment was higher, and the method was as follows: 1) 0.1 g uterine tissue was homogenized by adding 3 ml 0.1 M PBS buffer, centrifuged to collect the supernatant, and then 3 mL McFarland buffer was added to repeat the above steps to collect the supernatant; 2) 3 mL of n-hexane was added to mix and defat, and the supernatant was collected by centrifugation; 3) The matrix was obtained for matrix stability. The pretreatment method was combined with the previous high-performance liquid chromatography method for uterine A24 detection. By Figure 3 It can be seen that according to the established HPLC method for determining uterine stroma, impurities appeared at 1.17 min and 7.68 min, and the antimicrobial peptide A24 peaked at around 11 min, with no interference from other peaks nearby, indicating that the pretreatment method and HPLC method can be used for the detection of A24 in uterine tissue. (Note: The liquid phase detection results can clearly distinguish the target peak A24, and the peak value is the default output of the instrument and does not affect the observation of the results)

[0058] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0059] References: 1. ZASLOFF M., 1987. Magainins, a class of antimicrobial peptides from xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proceedings of the National Academy ofSciences, 84(15):5449-53. DOI: 10.1073 / pnas.84.15.5449.

[0060] 2. ALEXANDRE P, ZAVASCK I, 2014. Polymyxins for the treatment ofextensively-drug-resistant gram-negative bacteria: from pharmacokinetics tobedside. Expert Review of Anti Infective Therapy,12(5):531-533. DOI: 10.1586 / 14787210.2014.902307.

[0061] 3. KANTHAWONG S, BOLSCHER J, VEERMAN E, MARLE J, NAZMI K,WONGRATANACHEEWIN S, TAWEECHAISUPAPONG S, 2010. Antimicrobial activities ofLL-37 and its truncated variants against burkholderia thailandensis.International Journal of Antimicrobial Agents, 36(5): 447-452. DOI: 10.1016 / j.ijantimicag.2010.06.031.

[0062] 4. ZHENG X, YANG N, MAO R, HAO Y, TENG D, WANG J, 2022.Pharmacokinetics and pharmacodynamics of fungal defensin NZX againstStaphylococcus aureus-Induced Mouse Peritonitis Model. Frontiers inMicrobiology, 13: 865774. DOI: 10.3389 / fmicb.2022.865774.

[0063] 5. SCHNEIDER T, KRUSE T, WIMMER R, WIEDEMANN I, KRISTENSEN H, 2010.Plectasin, a fungal defensin, targets the bacterial cell wall precursor lipidII. Science, 328(5982): 1168-1172. DOI: 10.1126 / science.1185723.

Claims

1. A high performance liquid chromatography detection method for antimicrobial peptide A24, characterized in that: Detecting antimicrobial peptide A24 in liquid samples or tissue samples using high performance liquid chromatography; The liquid sample includes aqueous solution and plasma; the tissue sample includes uterine tissue; The method comprises the following steps: (1) The sample is pre-treated to obtain the sample to be tested; (2) Prepare standard sample solutions of antimicrobial peptide A24 of different concentrations, perform high performance liquid chromatography detection, and then draw a standard curve with the concentration of antimicrobial peptide A24 as the horizontal axis and the peak area as the vertical axis; (3) The sample to be tested is subjected to high performance liquid chromatography detection under the same conditions as step (2), and the content of antimicrobial peptide A24 in the sample to be tested is obtained according to the standard curve.

2. The method according to claim 1, characterized in that: The chromatograph used for high performance liquid chromatography detection is Agilent 1260 HPLC, UV detector, ZORBAX Eclipsc Plus C18 chromatographic column, size 4.6 mm × 250 mm, 5 μm, acidic organic solvent as mobile phase A, acidic aqueous solution as mobile phase B, gradient elution; column temperature 25-40 ° C; detection wavelength 220-280 nm; loading speed 0.5-1.5 mL / min.

3. The method according to claim 2, characterized in that The acidic aqueous solution is selected from at least one of trifluoroacetic acid, acetic acid, phosphoric acid, hydrochloric acid, and formic acid aqueous solutions, preferably a 0.01% to 1% trifluoroacetic acid aqueous solution, and more preferably a 0.1% trifluoroacetic acid aqueous solution.

4. The method according to claim 2, characterized in that: The organic solvent is selected from at least one of acetonitrile, methanol, ethanol and isopropanol, preferably acetonitrile; More preferably, the acidic organic solvent is acetonitrile containing 0.1% trifluoroacetic acid.

5. The method according to claim 4, wherein the elution gradient of mobile phase A is 10%-40%, and the elution gradient of mobile phase B is 90%-60%.

6. The method according to claim 5, characterized in that The gradient elution conditions were as follows: Wherein, the phase A is acetonitrile containing 0.1% trifluoroacetic acid, and the phase B is 0.1% trifluoroacetic acid aqueous solution.

7. The method according to claim 1, characterized in that The pretreatment method of the plasma sample in step (1) includes: adding 1% trifluoroacetic acid aqueous solution to the plasma, filtering through a 0.22 µm filter membrane, and preparing the sample to be tested.

8. The method according to claim 1, characterized in that: The pretreatment method of the uterine tissue sample in step (1) includes: adding 3 mL of 0.1 M PBS buffer to 0.1 g of uterine tissue sample, homogenizing and mixing, adding 3-5 mL of extracting solution, vortexing and mixing, and centrifuging to collect the supernatant; adding an equal volume of n-hexane to the supernatant, vortexing and mixing, and centrifuging to collect the lower layer of liquid; then purifying the sample with a purification column, concentrating the purified sample, and re-dissolving it with ultrapure water as a sample to be tested; The extract is prepared by mixing 1L of 0.1% formic acid aqueous solution with 400mL of methanol.

9. The method according to claim 8, characterized in that The purification column is selected from a WCX purification column, a MAX purification column, an MHA purification column or an HLB purification column, preferably an HLB purification column.

10. The method according to claim 9, characterized in that Purification and concentration methods include: 1) Activate the HLB purification column with methanol, ultrapure water or ammoniated methanol, preferably methanol; 2) Use deionized water or McFarland buffer for balance, preferably McFarland buffer; 3) eluting with a mixed solvent of acetonitrile and water, wherein the content of acetonitrile is 25% to 100%, preferably 40%; 4) The obtained eluate is concentrated and then reconstituted with ultrapure water to serve as the sample to be tested; The concentration was carried out using a constant temperature water bath nitrogen blowing apparatus with a nitrogen blowing temperature of 40±5°C and a time of 30 to 50 minutes.

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