Extraction method and detection method of antibacterial peptide NZ2114 in plasma

Abstract

The invention relates to the technical field of analysis and detection, in particular to an extraction method and a detection method of antibacterial peptide NZ2114 in plasma. Based on the physicochemical properties of the antibacterial peptide NZ2114 and the complexity of a plasma sample, the extraction method of the antibacterial peptide NZ2114 in the plasma is developed, and based on the LC-QTOF-MS / MS technology, the quantitative analysis and detection method of the antibacterial peptide NZ2114 in the plasma, which is low in detection limit, high in sensitivity, high in accuracy, high in precision, high in specificity, simple, convenient and rapid, is established. Accurate quantification of the antibacterial peptide NZ2114 in a complex matrix is achieved, pharmacokinetic research of the antibacterial peptide NZ2114 is promoted, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly to an extraction method and a detection method for antibacterial peptide NZ2114 in plasma. Background Art

[0002] Macromolecular polypeptide substances - antibacterial peptides isolated from organisms such as insects and mammals have characteristics such as high-efficiency sterilization, low drug resistance, and no residue. Antibacterial peptide NZ2114 is a derivative peptide obtained by amino acid modification of the first fungal defensin Plectasin, which has antibacterial specificity against Gram-positive bacteria and its antibacterial activity is superior to that of the parent peptide. In addition, NZ2114 has advantages such as intracellular bactericidal activity, no hemolytic activity, low cytotoxicity, and a long drug half-life, and is expected to become a new type of antibacterial drug. However, the inability to quantitatively detect antibacterial peptide NZ2114 after in vivo metabolism has always been a technical bottleneck restricting its clinical application.

[0003] Currently, the detection methods for antibacterial peptides mainly include biological assay, immunological method, in vivo imaging technology, chromatography, etc. The biological assay is a detection method based on functional specific biological responses to determine the biological activity of the substance to be tested, and is mainly used for the determination of the purity, stability, and potency of biological products. However, this method is easily affected by active metabolites, biological matrices, and environmental factors, and has a relatively high detection limit, unable to meet the needs of trace detection. In addition, the currently widely used immunological method is mainly ELISA. This technology mainly relies on the binding of antigens and antibodies, so it is easily interfered by substances such as endogenous enzymes and proteins, resulting in cross-reactions and even false positives, and the specificity of this method is relatively poor. Therefore, this method is not suitable for detecting endogenous complex samples such as plasma. The in vivo imaging technology mainly realizes the qualitative detection of drugs in animals by fluorescently labeling the target substance, and is an effective means for drug in vivo tracing. Therefore, this technology can be used to explore the metabolism of new drugs in animals. Chromatography technology has been widely used in the research of fields such as medicine, chemistry, environment, and agronomy due to its advantages of high resolution, wide applicability, strong specificity, and low detection limit. Liquid chromatography-mass spectrometry (LC-MS) technology combines a chromatograph with a mass spectrometer in series. The mass spectrometer distinguishes chemically or biologically similar proteins and peptides (intact or peptide fragments) according to their different mass-to-charge ratios, improving the analysis sensitivity and specificity for the target substance. In addition, LC-MS uses ion fragments as marker signals, has a higher response to the target substance, and a lower detection limit.

[0004] Currently, there are few analytical methods for detecting polypeptide substances such as antibacterial peptides using high-performance liquid chromatography technology. In the prior art, CN117534742A discloses an extraction, purification, and detection method for antibacterial peptide NZ2114 in feed. This method targets the antibacterial peptide NZ2114 in feed and is not applicable to the detection of antibacterial peptide NZ2114 in plasma.

[0005] Therefore, how to provide an analytical detection method for quantitatively detecting the antimicrobial peptide NZ2114 in plasma has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address these technical challenges, the present invention established a method for detecting the antimicrobial peptide NZ2114 in plasma samples using liquid chromatography-mass spectrometry (LC-QTOF-MS / MS), enabling quantitative detection of the antimicrobial peptide NZ2114 in plasma. Based on this, the following technical solution is proposed.

[0007] First, the present invention provides a method for extracting antimicrobial peptide NZ2114 from plasma, comprising: mixing a plasma sample with an extracting solution, then adding a first reducing agent and incubating the mixture, then adding a second reducing agent and allowing the mixture to react, and obtaining a first supernatant after the reaction; adding the extracting solution to the precipitate after the reaction and reacting the mixture, and obtaining a second supernatant after the reaction; and combining the first supernatant and the second supernatant to obtain an extract of the antimicrobial peptide NZ2114; The extracting solution is an acetonitrile solution containing formic acid; the first reducing agent is a DL-dithiothreitol solution; and the second reducing agent is a mixed solution of 2-iodoacetamide and triethylammonium bicarbonate buffer (TEAB).

[0008] The use of the above extract in combination with the first reducing agent and the second reducing agent can remove non-target substances in plasma as much as possible through protein precipitation, with good purification effect, which is conducive to the subsequent quantitative analysis of the antimicrobial peptide NZ2114 by liquid chromatography-mass spectrometry.

[0009] Preferably, in the extract, the volume percentage of formic acid is 0.05% to 0.2%, and the volume percentage of acetonitrile is 75% to 85%.

[0010] Preferably, the final concentration of DL-dithiothreitol after adding the first reducing agent is 0.5-2 mM, more preferably 0.8-1.2 mM.

[0011] Preferably, the final concentration of 2-iodoacetamide after adding the second reducing agent is 2-5 mM.

[0012] Preferably, the incubation temperature is 35° C. to 40° C.; and / or the incubation time is more than 1 hour.

[0013] Preferably, the volume ratio of the plasma sample to the extract is 1:(2-5).

[0014] Preferably, the plasma sample contains the antimicrobial peptide NZ2114.

[0015] Preferably, the plasma sample is derived from experimental animals treated with the antimicrobial peptide NZ2114 solution by oral gavage.

[0016] Preferably, the conditions for the static reaction are to avoid light at room temperature.

[0017] More preferably, the extraction method includes: mixing the plasma sample with the extraction solution, vortexing, then adding the first reducing agent and incubating (preferably for more than 1 h) at (preferably 35°C - 40°C), adding the second reducing agent and shaking well, placing in the dark at room temperature (preferably for more than 30 min), mixing well and then carrying out a static reaction (preferably for 5 - 10 min), centrifuging to obtain the first supernatant; adding the extraction solution to the precipitate after centrifugation, ultrasonically mixing and then carrying out a static reaction (preferably for 5 - 10 min), centrifuging to obtain the second supernatant, and combining the first supernatant and the second supernatant to obtain the antimicrobial peptide NZ2114 extraction solution; The extraction solution is an acetonitrile solution containing formic acid; the first reducing agent is a DL-dithiothreitol solution; the second reducing agent is a mixed solution of 2-iodoacetamide and triethylammonium bicarbonate buffer solution.

[0018] Preferably, the centrifugation speed is above 10000 rpm and the time is above 5 min.

[0019] Preferably, after combining the first supernatant and the second supernatant, centrifugation (preferably centrifuging at 140000 rpm for more than 5 min) is carried out to obtain the antimicrobial peptide NZ2114 extraction solution.

[0020] Furthermore, the present invention provides a method for detecting the antimicrobial peptide NZ2114 in plasma, including: after obtaining the antimicrobial peptide NZ2114 extraction solution by using the extraction method described in any one of the above, detecting by using the LC-QTOF-MS / MS technology.

[0021] Preferably, after mixing the antimicrobial peptide NZ2114 extraction solution with ultrapure water at a volume ratio of 1:(2 - 5), detecting by using the LC-QTOF-MS / MS technology.

[0022] Preferably, in the LC-QTOF-MS / MS technology, mobile phase A is water, mobile phase B is acetonitrile, and the chromatographic mobile phase elution gradient is as follows:

[0023] During the chromatographic mobile phase elution process, the sum of the volume percentages of mobile phase A and mobile phase B is 100%.

[0024] Preferably, the chromatographic column model is ACQUITY UPLC BEH C8 Column, with specifications of 130 Å, 1.7 µm, 2.1 mm×100 mm; and / or, the column temperature is 40°C - 45°C; and / or, the flow rate is 0.2 - 0.3 mL / min.

[0025] Preferably, in the LC-QTOF-MS / MS technique, the mass spectrometry conditions include: parallel reaction monitoring is adopted, the ion source is in the electrospray detection mode, the curtain gas is 30-40 psi, the nebulizing gas is 45-55 psi, the auxiliary heater is 45-55 psi, the deionized spray voltage is 5200-5800 V, and the ion source temperature is 420-480 °C.

[0026] Preferably, the concentration of antibacterial peptide NZ2114 is obtained by establishing a standard curve using the internal standard method or the external standard method.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: Based on the physicochemical properties of antibacterial peptide NZ2114 and the complexity of plasma samples, the present invention has developed an extraction method for antibacterial peptide NZ2114 in plasma, and has established a quantitative analysis and detection method for antibacterial peptide NZ2114 in plasma with low detection limit, high sensitivity, high accuracy, high precision, strong specificity and simplicity and rapidity based on the LC-QTOF-MS / MS technique, realizing the accurate quantification of antibacterial peptide NZ2114 in complex matrices, promoting the pharmacokinetic research of antibacterial peptide NZ2114, and having broad application prospects. Description of the Drawings

[0028] Figure 1 It is the chromatogram of the processed blank matrix detected by the detection method of the present invention.

[0029] Figure 2 It is the chromatogram of the 1080 ng / mL NZ2114 standard solution detected by the detection method of the present invention.

[0030] Figure 3 It is the chromatogram of the processed blank matrix added with 1080 ng / mL NZ2114 standard solution detected by the detection method of the present invention.

[0031] Figure 4 It is the chromatogram of the biological sample detected at 1 h after intragastric administration by the detection method of the present invention.

[0032] Figure 5 It is the matrix correction standard curve of the detection method of the present invention.

[0033] Figure 6 It is the concentration-time curve. Detailed Embodiments

[0034] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts fall within the protection scope of the present invention.

[0035] In the embodiments provided in this specification, for those without specific technical or conditions indicated, they shall be in accordance with the technologies or conditions described in the literature in the field or in accordance with the product specifications. For reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained through regular channels.

[0036] Example 1 This example provides a method for detecting antibacterial peptide NZ2114 in plasma, and the steps are as follows: 1. Main reagents NZ2114 standard (purity > 99%, Institute of Botany, Chinese Academy of Sciences), acetonitrile (chromatographic grade) was purchased from Thermo Fisher Scientific (China) Co., Ltd.; ammonia water (chromatographic grade, ≥ 98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; DL-dithiothreitol (analytical pure, > 99.5%), 2-iodoacetamide (NRM, ≥ 99%), and triethylammonium bicarbonate buffer were all purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; distilled water (Watsons).

[0037] 2. Main instruments Triple quadrupole time-of-flight liquid chromatography-mass spectrometry (Triple Tof 6600) was purchased from AB SCIEX; Vortex-Genie2 was purchased from Scientific Industries; electric thermostatic water bath (DZKW-5-4) was purchased from Yongguangming Medical Instrument Co., Ltd.; analytical balance (ME204T) was purchased from Mettler Toledo Instruments (Shanghai) Co., Ltd.; centrifuge (5810R) was purchased from Eppendorf AG, Germany; ultrasonic instrument (KQ-500DE) was purchased from Kunshan Ultrasonic Instrument Co., Ltd.; pipette (2.5 - 1000 μL) was purchased from Eppendorf AG, Germany.

[0038] 3. Preparation of standard stock solution Accurately weigh 20 mg of NZ2114 standard and dissolve it in 2 mL of ultrapure water to prepare a 10 mg / mL standard stock solution, aliquot it into 1.5 mL cryotubes, and store it at -20 °C for later use.

[0039] 4. Preparation of plasma samples 4.1 Blank plasma samples Take female SPF mice weighing about 20 g that have fasted for 24 h and have not been injected with NZ2114 solution. Use sterilized forceps to remove one eyeball, and drip the blood into an EP tube containing 2% sodium heparin. After sample collection, invert the EP tube up and down to allow the blood to fully contact the sodium heparin. Let it stand still at 4 °C for 20 - 30 min, then centrifuge at 5000 rpm for 10 min. Transfer the plasma to a clean EP tube to obtain blank plasma samples.

[0040] 4.2 Plasma samples to be measured Take the same female mice that have fasted for 12 h (with free access to water during this period) and are intragastrically injected with NZ2114 solution at a concentration of 1 g / kg. The intragastric administration volume is 0.5 mL, and the dose is 1 g / kg NZ2114 solution. At 0.25, 0.5, 1, 2, 3, 3.5, 4, 4.5, 5, 6, 12, and 24 h after administration, collect blood samples at each time point by eye bleeding, drip them into an EP tube containing 2% sodium heparin, invert the tube up and down to allow the blood to fully contact the sodium heparin. After standing still at 4 °C for 20 - 30 min, centrifuge at 5000 rpm for 10 min, and transfer the supernatant to a clean EP tube to obtain plasma samples. Set 3 replicates for each time point. Take 100 μL of the plasma samples at each time point and add DL-dithiothreitol with a final concentration of 1 mM for detection.

[0041] 5. Pretreatment Add 100 μL of blank plasma to 50 μL of NZ2114 standard solutions with a series of different concentrations. After thorough mixing, add 350 μL of 80% acetonitrile - 1% formic acid aqueous extraction solution (when measuring 100 μL of sample plasma, directly mix it with the extraction solution). Vortex and shake, then add DL-dithiothreitol to a final concentration of 1 mM, shake and mix well, and incubate in a 37 °C water bath for 1 h. Add 1 μL of 2-iodoacetamide prepared with triethylammonium bicarbonate buffer as the solvent to the incubated solution to a final concentration of 3 mM. Shake and mix well, place it in the dark at room temperature for 30 min; take it out, mix it up and down, let it stand and react for 5 - 10 min, centrifuge at 10000 rpm for 5 min, take the supernatant, add 500 μL of 80% acetonitrile - 1% formic acid aqueous extraction solution to the precipitate, sonicate for 2 min to fully mix the precipitate and the extraction solution, let it stand and react for 5 - 10 min, centrifuge at 10000 rpm for 5 min, take the supernatant, combine the two supernatants, centrifuge at 14000 rpm for 5 min, take 200 μL of the supernatant and add it to an injection vial, then add 600 μL of ultrapure water, mix well and set aside for use.

[0042] 6. LC-QTOF-MS / MS detection conditions 6.1 Chromatographic conditions Chromatographic column: ACQUITY UPLC BEH C8 Column, 130 Å, 1.7 µm, 2.1 mm×100 mm; Mobile phase A: 100% ultrapure water; Mobile phase B: 100% acetonitrile; Column temperature: 45 °C; Flow rate: 0.3 mL / min; Injection volume: 3 μL; The elution gradient of the chromatographic mobile phase is shown in Table 1.

[0043] Table 1 Elution gradient of the chromatographic mobile phase

[0044] 6.2 Mass spectrometry conditions Parallel reaction monitoring (PRM) was adopted, with the electrospray (ES) detection mode of the ion source, curtain gas (CUR) 35 psi, nebulizing gas (GS1) 50 psi, auxiliary heater (GS2) 50 psi, deionized spray voltage (ISV) 5500 V, and ion source temperature (TEM) 450 °C.

[0045] Using the above-mentioned pretreatment and LC-QTOF-MS / MS detection conditions, the specificity, linear relationship, detection limit, quantitation limit, recovery rate, precision, and matrix effect of the detection method were investigated.

[0046] Example 2 In this example, the mass spectrometry precursor ions of antibacterial peptide NZ2114 were determined as follows: Antibacterial peptide NZ2114 was ionized by electrospray ionization (ESI) in the positive ion mode to obtain a large number of ion fragments. After scanning by the mass analyzer and ion detector (parallel reaction monitoring, PRM scanning mode), a high-resolution mass spectrometry map of the target peptide segment was collected. Precursor ions with relatively high signal intensities were screened. Three precursor ions with relatively strong signal intensities could be seen from the first-order mass spectrometry map, which were 735.98 m / z ([M+6H]+), 883.03 m / z ([M+5H]+), and 1103.54 m / z ([M+4H]+) respectively. Two precursor ions with high-intensity signals of 735.98 m / z ([M+6H]+) and 883.03 m / z ([M+5H]+) were also screened from the second-order mass spectrometry map. Finally, 735.98 m / z ([M+6H]+) and 883.03 m / z ([M+5H]+) were used for the qualitative and quantitative detection of antibacterial peptide NZ2114.

[0047] Example 3 In this example, the specificity of the detection method was investigated as follows: A certain concentration of NZ2114 standard solution was added to the blank plasma sample to make its final concentration 1080 ng / mL, thus obtaining a 1080 ng / mL NZ2114 standard sample; then a certain volume of standard solution was added to the blank matrix treated by the pretreatment method of Example 1 to make its final concentration 1080 ng / mL, obtaining a blank matrix plus standard sample; finally, a 20 g female SPF mouse was taken, intragastrically injected with 0.5 mL of 1 g / kg antimicrobial peptide NZ2114, blood was taken from the eyeball 1 h later, plasma was taken after centrifugation at 5000 rpm for 10 min, and then detection was carried out according to the detection method of Example 1.

[0048] The detection results are as Figures 1 to 4 shown. The detection results of the treated blank matrix are as Figure 1 shown, Figure 2 showing that the peak emergence time of antimicrobial peptide NZ2114 is 3.5 min, Figure 3 it can be seen from the chromatogram that there are fewer impurity peaks in the chromatographic peaks before 5.3 min and they can be separated from the target peak, so the pretreatment method can effectively reduce the interference of endogenous components in plasma on antimicrobial peptide NZ2114. Figure 4 showing that the peak emergence time of antimicrobial peptide NZ2114 in the biological sample is also 3.5 min and there are no miscellaneous peaks around it, and specific detection of antimicrobial peptide NZ2114 in the biological sample can be achieved.

[0049] Example 4 In this example, the linear relationship of the detection method was investigated, and the steps are as follows: NZ2114 standard solution was added to the blank plasma sample to make its final concentrations 40 ng / mL, 120 ng / mL, 360 ng / mL, 1080 ng / mL, 3140 ng / mL and 9720 ng / mL respectively, and then detection was carried out according to the detection method of Example 1 to obtain the peak area.

[0050] A matrix correction standard curve was established with the added concentration of antimicrobial peptide NZ2114 as the abscissa and the corresponding peak area as the ordinate. The results are as Figure 5 shown. The linear equation is y = 227.79x - 14182, and the correlation coefficient R² is 0.9991, indicating that antimicrobial peptide NZ2114 has a good correlation in the concentration range of 40 - 9720 ng / mL.

[0051] Example 5 In this example, the detection limit and quantification limit of the detection method were investigated, and the steps are as follows: The test was carried out according to the steps of Example 4. When the added concentration was 40 ng / mL, accurate quantification of NZ2114 in plasma could be achieved, and S / N≥10 was satisfied. When the added concentration was 15 ng / mL, S / N≥3. Therefore, 15 ng / mL was taken as the detection limit for the instrument to detect antibacterial peptide NZ2114 in plasma, and 40 ng / mL was taken as the lower limit of quantification for this detection method.

[0052] Example 6 In this example, the recovery rate of the detection method was investigated, and the steps were as follows: A certain concentration of antibacterial peptide NZ2114 solution was added to the blank matrix to obtain low, medium, and high concentration matrix spiked samples with final concentrations of 40 ng / mL, 1080 ng / mL, and 9720 ng / mL respectively. The samples at the three concentration levels were detected according to the detection method of Example 1. The peak areas detected by liquid chromatography-mass spectrometry were substituted into the matrix correction standard curve of the current batch to calculate the actual concentration of antibacterial peptide NZ2114. The ratio of the actual concentration to the theoretical concentration was the added recovery rate. The results are shown in Table 2. The added recovery rates of low, medium, and high concentrations were 89.28%, 103.41%, and 94.91% respectively.

[0053] Table 2 Recovery rate test data

[0054] Example 7 In this example, the precision of the detection method was investigated, and the steps were as follows: A certain concentration of antibacterial peptide NZ2114 solution was added to the pre-treated blank matrix to obtain low, medium, and high concentration matrix spiked samples with final concentrations of 40 ng / mL, 1080 ng / mL, and 9720 ng / mL respectively. The samples at the three concentration levels were detected according to the detection method of Example 1. The peak areas were detected on the instrument intra-day and inter-day respectively, substituted into the correction standard curve established in the current batch, and the actual concentration of antibacterial peptide NZ2114 was calculated, and the coefficient of variation (RSD / %) was calculated. The results are shown in Table 3. The intra-day relative errors of low, medium, and high concentration samples were 6.67%, 4.78%, and 2.75% respectively, and the inter-day relative errors were 7.7%, 6.3%, and 6.19% respectively, all lower than 15%. It shows that the precision of this method is good and meets the requirements of the relative standard error (RSD) lower than 15% or 20% in the quantitative analysis method of biological samples.

[0055] Table 3 Precision test data

[0056] Example 8 In this example, the matrix effect of the detection method was investigated, and the steps were as follows: Using the pre-treated blank matrix and acetonitrile as solvents respectively, add antibacterial peptide NZ2114 solutions at certain concentrations to make their final concentrations 40 ng / mL, 1080 ng / mL, and 9720 ng / mL respectively, to obtain matrix-spiked samples and organic-phase spiked samples after low, medium, and high concentration treatments. Detect them on the machine according to the detection method of Example 1 to obtain the peak area of antibacterial peptide NZ2114. The ratio of the latter to the former peak area is the matrix effect value of each concentration sample. The results are shown in Table 4, indicating that there is a certain matrix inhibition effect in low, medium, and high concentration samples, especially in low concentration samples. Therefore, a matrix-corrected standard curve should be used for correction during the determination of biological samples.

[0057] Table 4 Matrix effect test data

[0058] In summary, the present invention has established an LC-QTOF / MS / MS detection method for antibacterial peptide NZ2114 in mouse plasma, and has carried out methodological verification on this method, confirming the feasibility of this method.

[0059] Example 9 In this example, the detection method of Example 1 was used to detect antibacterial peptide NZ2114 in the plasma of mice administered by gavage, and the steps are as follows: After the mice were fasted for 12 h (drinking water freely during this period), the mice were gavaged with 0.5 mL of antibacterial peptide NZ2114 solution at a concentration of 1 g / kg. Blood samples were obtained by eye bleeding at 0.25, 0.5, 1, 2, 3, 3.5, 4, 4.5, 5, 6, 12, and 24 h after administration, dropped into EP tubes containing 2% sodium heparin, and inverted up and down to make the blood fully contact with sodium heparin. After standing at 4 °C for 20 - 30 min, centrifuged at 5000 rpm for 10 min, and the supernatant was transferred to a clean EP tube to obtain plasma samples at each time point, with 3 replicates set at each time point. 100 μL of plasma samples at each time point were taken respectively, and DL-dithiothreitol with a final concentration of 1 mM was added to obtain the biological samples to be detected, and the detection method of Example 1 was used for detection to obtain the blood drug concentration. Using Graphpad Prism software, a blood drug concentration-time curve was plotted with the administration time as the abscissa and the blood drug concentration as the ordinate, as Figure 6 shown.

[0060] It can be seen that after the 1 g / kg antibacterial peptide NZ2114 solution was administered to mice by gavage, the drug showed a trend of first decreasing and then increasing twice in their bodies, and the drug-time curve finally tended to be stable. From the drug-time curve, it can be seen that the metabolic times of 4.5 h and 6 h are the two peaks respectively, so the drug-time curve shows a bimodal shape.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for extracting antibacterial peptide NZ2114 from plasma, characterized in that, Comprising: Mix the plasma sample with the extraction solution, then add the first reducing agent and incubate, then add the second reducing agent and let it stand for reaction, and obtain the first supernatant after standing for reaction; add the extraction solution to the precipitate after reaction and react to obtain the second supernatant, and combine the first supernatant and the second supernatant to obtain the extraction solution of antibacterial peptide NZ2114; The extraction solution is an acetonitrile solution containing formic acid; the first reducing agent is a DL-dithiothreitol solution; the second reducing agent is a mixed solution of 2-iodoacetamide and triethylammonium bicarbonate buffer solution.

2. The extraction method according to claim 1, wherein In the extraction solution, the volume percentage of formic acid is 0.05% - 0.2%, and the volume percentage of acetonitrile is 75% - 85%.

3. The extraction method according to claim 1, characterized in that, The final concentration of DL-dithiothreitol after adding the first reducing agent is 0.5 - 2 mM; And / or, the final concentration of 2-iodoacetamide after adding the second reducing agent is 2 - 5 mM.

4. The extraction method according to claim 1, characterized in that The incubation temperature is 35°C - 40°C; and / or, the incubation time is more than 1 h.

5. The extraction method according to claim 1, wherein The volume ratio of the plasma sample to the extraction solution is 1:(2 - 5).

6. A method for detecting antibacterial peptide NZ2114 in plasma, characterized in that, Comprising: After obtaining the extraction solution of antibacterial peptide NZ2114 by using the extraction method described in any one of claims 1 - 5, detect it by LC-QTOF-MS / MS technology.

7. The detection method according to claim 6, characterized in that, In the LC-QTOF-MS / MS technology, mobile phase A is water, mobile phase B is acetonitrile, and the chromatographic mobile phase elution gradient is as follows: During the elution of the chromatographic mobile phase, the sum of the volume percentages of mobile phase A and mobile phase B is 100%.

8. The detection method according to claim 7, wherein The chromatographic column model is ACQUITY UPLC BEH C8Column, its specifications are 130 Å, 1.7 µm, 2.1 mm×100 mm; and / or, the column temperature is 40°C - 45°C; and / or, the flow rate is 0.2 - 0.3 mL / min.

9. The detection method according to claim 6, characterized in that, In the LC-QTOF-MS / MS technology, the mass spectrometry conditions include: using parallel reaction monitoring, electrospray ionization detection mode, curtain gas 30 - 40 psi, nebulizer gas 45 - 55 psi, auxiliary heater 45 - 55 psi, deionized spray voltage 5200 - 5800 V, ion source temperature 420 - 480 °C.

10. The detection method according to any one of claims 6 to 9, characterized in that Establish a standard curve by the internal standard method or the external standard method to obtain the concentration of antibacterial peptide NZ2114.

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