Method for detecting blood concentration of elboviridecane through mass spectrum method

Abstract

The invention provides a method for detecting the blood concentration of an ibovirideai long-acting inhibitor through a mass spectrometry method. Specifically, the method provided by the invention comprises the following steps: (Z1) providing a plasma sample containing the ibovipentam; (Z2) carrying out enzymolysis on the sample by using protease to obtain a sample containing characteristic peptide; and (Z3) detecting the content of the characteristic peptide, and calculating according to the content of the characteristic peptide to obtain the concentration of the elbovipentam in the sample. The method provided by the invention has the advantages of strong specificity, high accuracy and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for detecting the blood concentration of Abovirtadine (ABT) by mass spectrometry. Background Art

[0002] Human immunodeficiency virus (HIV) is the virus that causes acquired immune deficiency syndrome (AIDS), which has become one of the most serious health problems in the world. HIV belongs to a class of viruses called retroviruses. The HIV-1 envelope glycoprotein 120 (gp120) complex consists of three receptor-binding gp120 subunits and three fusion protein gp41 subunits, which enable fusion between the HIV viral envelope and the target cell membrane through interactions with sequences of CD4 glycoprotein and chemokine receptors on the cell surface, and provides an attractive target for the development of antiviral agents. During the fusion reaction, the N-terminal and C-terminal heptad repeat regions (NHR and CHR) of the gp41 extracellular domain refold into a stable six-helix bundle structure (6-HB), presenting a fusion-active conformation that can bridge the viral and cell membranes for fusion. Many peptides derived from gp41 CHR (C-peptides) can specifically inhibit viral entry at low nanomolar concentrations, including abavirtide, a maleimidopropionic acid (MPA)-modified peptide HIV fusion inhibitor. Abavirtide can be irreversibly conjugated to serum albumin. Earlier studies have demonstrated its long in vivo half-life and potent anti-HIV activity.

[0003] Abovirtide is a chemically synthesized polypeptide consisting of 34 amino acids with an N-{2-[2-(N-(3-maleimidepropionyl)amino)ethoxy]ethoxy}acetyl group linked to the ε-amino group of lysine at position 13. Its chemical name is: acetyl-Glu-Glu-Glu-Glu-Glu-Glu-Glu-Gly-Isoleucine-Tyr-Thyr-(N-{2-[2-(N-(3-maleimidepropionyl)amino)ethoxy]ethoxy}acetyl)lys-Leu-Isoleucine-Tyr-Th ... 204 H 306 N 54 O 72 , and has a molecular weight of 4666.93, and has the following chemical structure:

[0004]

[0005] Abovirtide forms a conjugate with serum albumin in vivo. Although there are currently a variety of methods for detecting blood drug concentrations, such as traditional ELISA, MSD (Meso Scale Discovery, electrochemiluminescence detection technology) and tandem hybrid four-rod time-of-flight (Q-TOF) mass spectrometer detection. However, due to the large molecular weight of the Abovirtide-serum albumin conjugate, the Q-TOF has low detection sensitivity for it, and the ELISA and MSD detection sensitivities are also limited, making it difficult to detect low blood drug concentrations of peptide conjugates at longer time points after administration in pharmacokinetic (PK) tests, and it is impossible to accurately determine its half-life. Moreover, after binding, Abovirtide is also difficult to completely dissociate from the conjugated molecule, which makes it difficult to detect the complete molecule of Abovirtide in plasma samples.

[0006] Therefore, there is an urgent need in the art to find a method that can efficiently and accurately measure the blood concentration of elbovirtide. Summary of the invention

[0007] The present invention provides a method for efficiently and accurately measuring the blood concentration of ibaviridine.

[0008] In a first aspect of the present invention, a method for non-diagnostic and non-therapeutic detection of the content of ibovavirtide polypeptide in a sample is provided, comprising the steps of:

[0009] (Z1) providing a sample containing the elbovirtide;

[0010] (Z2) enzymatically hydrolyzing the sample using a protease to obtain a sample containing characteristic peptides; and

[0011] (Z3) detecting the content of the characteristic peptide, and calculating the concentration of ibovavir in the sample based on the content of the characteristic peptide;

[0012] Wherein, the ibovavirtide is a polypeptide that can be enzymatically hydrolyzed to form a characteristic peptide, and the protease is an enzyme that can enzymatically hydrolyze ibovavirtide to obtain a characteristic peptide.

[0013] In another preferred embodiment, the characteristic peptide has an amino acid sequence with more than 85% homology to the sequence shown in SEQ ID NO:1.

[0014] In another preferred example, the amino acid sequence of ibovavirtide is as shown in SEQ ID NO: 2.

[0015] In another preferred embodiment, in the ibovavirtide, position 13 of SEQ ID NO: 2 is modified with N-{2-[2-(N-(3-maleimidopropionyl)amino)ethoxy]ethoxy}acetyl.

[0016] In another preferred embodiment, the protease is trypsin.

[0017] In another preferred embodiment, in step (Z3), the content of the enzymatically hydrolyzed characteristic peptide is detected by liquid chromatography-mass spectrometry (LCMS).

[0018] In another preferred embodiment, the liquid chromatography-mass spectrometry (LCMS) can be a triple tandem quadrupole mass spectrometry (QQQMS), a tandem quadrupole linear ion trap mass spectrometry (Q-TRAP), a quadrupole time-of-flight mass spectrometry (Q-TOF), an ion trap-time-of-flight mass spectrometry (IT-TOF), etc.

[0019] In another preferred embodiment, the liquid chromatography-mass spectrometry (LCMS) is a triple tandem quadrupole mass spectrometry (QQQMS).

[0020] In another preferred embodiment, the sample is selected from the following group: biological samples, environmental samples, product sampling; preferably, the sample is a biological sample; more preferably, the sample is a blood sample.

[0021] In a second aspect of the present invention, a method for non-diagnostic and non-therapeutic analysis of the half-life of elbovirtide in a blood sample is provided, characterized in that the method comprises the steps of:

[0022] (S1) providing a blood sample;

[0023] (S2) mixing the ibovavirtide with the blood sample, so that the ibovavirtide reacts with the plasma albumin in the blood sample;

[0024] (S3) enzymatically hydrolyzing the sample using a protease to obtain a sample containing enzymatically hydrolyzed characteristic peptides; and

[0025] (S4) detecting the content of the enzymatically hydrolyzed characteristic peptide, calculating the concentration of ibovavir in the blood sample based on the content of the characteristic peptide, and calculating the half-life of ibovavir based on the concentration;

[0026] Wherein, the abovirtide is a polypeptide that can be enzymatically hydrolyzed to form a characteristic peptide, and the protease is an enzyme that can enzymatically hydrolyze abovirtide to obtain a characteristic peptide.

[0027] In another preferred embodiment, the enzymatic characteristic peptide has an amino acid sequence with more than 85% homology to the sequence shown in SEQ ID NO:1.

[0028] In another preferred example, the amino acid sequence of ibovavirtide is as shown in SEQ ID NO: 2.

[0029] In another preferred embodiment, in the ibovavirtide, position 13 of SEQ ID NO: 2 is modified with N-{2-[2-(N-(3-maleimidopropionyl)amino)ethoxy]ethoxy}acetyl.

[0030] In another preferred embodiment, in the method, the step (S4) further includes: continuously detecting the content of the enzymatic characteristic peptide, and then drawing a change curve according to the change of the concentration of the ibovavirtide detected at different times, so as to obtain the drug-time curve of the enzymatic characteristic peptide, and calculating the half-life of the ibovavirtide based on the drug-time curve.

[0031] In another preferred embodiment, in step (S4), the content of the characteristic peptide is determined by liquid chromatography-mass spectrometry (LCMS).

[0032] In another preferred embodiment, the liquid chromatography-mass spectrometry (LCMS) may be a triple tandem quadrupole mass spectrometer (QQQMS), a tandem quadrupole linear ion trap mass spectrometer (Q-TRAP), a quadrupole time-of-flight mass spectrometer (Q-TOF), an ion trap-time-of-flight mass spectrometer (IT-TOF), preferably a triple tandem quadrupole mass spectrometer (QQQMS).

[0033] In another preferred embodiment, the liquid chromatography-mass spectrometry (LCMS) is a triple tandem quadrupole mass spectrometry (QQQMS).

[0034] In another preferred embodiment, the reaction time of step (S2) is 5-60 minutes.

[0035] In another preferred embodiment, in step (Z3), the protease is trypsin.

[0036] In a third aspect of the present invention, a kit is provided, the kit comprising a characteristic peptide having an amino acid sequence having 85% homology to the sequence shown in SEQ ID NO:1.

[0037] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A typical chromatogram of Elbovirtide assayed by competitive ELISA is shown.

[0039] Figure 2 The ABT blood concentration test obtained in Example 2 is shown.

[0040] Figure 3The ABT blood drug concentration test calibration curve obtained in Example 2 is shown. DETAILED DESCRIPTION

[0041] After extensive and in-depth research, the inventors unexpectedly discovered a method for more objectively and accurately measuring the concentration of ibovavirtide in blood. Specifically, the present invention uses the MRM mode of triple tandem quadrupole mass spectrometry (QQQMS) for quantitative detection of characteristic peptides of ibovavirtide, thereby detecting the concentration of ibovavirtide in blood. The present invention was completed on this basis.

[0042] the term

[0043] As used herein, the terms "Abovirtide" and "ABT" can be used interchangeably, and include an amino acid sequence such as the sequence shown in SEN ID NO: 2 (WEEWDREINNYTKLIHELIEESQNQQEKNEQELL). The sequence of SEQ ID NO: 2 can be modified, for example, in a preferred embodiment, the 13th amino acid of the above sequence can be modified by N-{2-[2-(N-(3-maleimidopropionyl)amino)ethoxy]ethoxy}acetyl.

[0044] As used herein, the term "protease" refers to a protease capable of enzymatically degrading Elbovirtide. In the present invention, the protease used is preferably trypsin.

[0045] As used herein, the terms "characteristic peptide" and "enzymatic characteristic peptide" can be used interchangeably and refer to the peptide segment formed after the tested ibovavirtide is enzymatically hydrolyzed by a protease. In a preferred embodiment of the present invention, the enzymatic fragment (i.e., the enzymatic fragment 29-34) formed after the ibovavirtide is hydrolyzed by trypsin is the characteristic enzymatic fragment of the tested ibovavirtide, and the fragment has no source in other species, therefore, it will not be interfered by the inherent components of conventional biological samples, such as blood samples, and can be used as a detection characteristic peptide.

[0046] In the present invention, "QQQ" refers to triple tandem quadrupole, and "QQQMS" refers to triple tandem quadrupole mass spectrometry.

[0047] The abbreviation "MRM" refers to Multiple Reaction Monitoring.

[0048] The abbreviation "PK" refers to pharmacokinetics, or pharmacokinetics for short.

[0049] Method for detecting the concentration of ibovavirtide-albumin conjugate in samples

[0050] The present invention provides a method for detecting the concentration of a conjugate of ibovavirtide and serum albumin in a sample, comprising the steps of:

[0051] (Z1) providing a sample containing the conjugate of elbovirtide and serum albumin;

[0052] (Z2) enzymatically hydrolyzing the sample using a protease to obtain an enzymatically hydrolyzed sample containing enzymatically hydrolyzed characteristic peptides; and

[0053] (Z3) detecting the content of the enzymatically hydrolyzed characteristic peptide, and calculating the concentration of the conjugate of ibovavirtide and serum albumin in the sample based on the content.

[0054] Wherein, the ibovavirtide is a polypeptide that can be coupled to albumin and enzymatically hydrolyzed to form the enzymatic characteristic peptide; and the enzymatic characteristic peptide has a sequence as shown in SEQ ID NO:1, or an amino acid sequence having more than 85% (preferably more than 90%, more preferably more than 95%, and most preferably more than 99%) homology with the sequence as shown in SEQ ID NO:1.

[0055] Since the complex formed by coupling Abovirtadine with albumin is a macromolecule, it cannot be used for detection by some conventional high-precision detection methods (such as mass spectrometry). In this regard, the inventor unexpectedly found that when a part of the polypeptide (such as Abovirtadine) is combined with a sample containing albumin (such as a blood sample), the generated complex can be enzymatically hydrolyzed by a protease, thereby generating a characteristic peptide shown in an amino acid sequence as shown in SEQ ID NO: 1 or having more than 85% homology (preferably more than 90%, more preferably more than 95%, and most preferably more than 99%) with the sequence shown in SEQ ID NO: 1. The inventor found that the enzymatic characteristic peptide sequence does not exist in other common species (including mice, rats, and humans), especially in commonly used blood samples. Therefore, it is a characteristic sequence of an albumin-polypeptide complex. By using a method with high precision and high specificity to detect it, the concentration of the albumin-polypeptide complex in the sample can be effectively detected.

[0056] In the present invention, the preferred protease is trypsin.

[0057] The method can be used to detect any polypeptide that can complex with albumin and be enzymatically hydrolyzed to form the characteristic sequence, which in a specific embodiment of the present invention is ibovavirtide; preferably, the ibovavirtide has SEQ ID NO: 2, and the 13th amino acid of SEQ ID NO: 2 is modified by N-{2-[2-(N-(3-maleimide propionyl)amino)ethoxy]ethoxy}acetyl.

[0058] In the above method, there is no particular limitation on the method for detecting the concentration of the enzymatic characteristic peptide, and methods known in the art for detecting the concentration of characteristic peptides or proteins can be used, such as competitive ELISA or MSD (electrochemiluminescence detection technology), etc. Since the sample concentration is low in some embodiments, a high-precision and high-specificity test method is required, so a preferred detection method is liquid chromatography-mass spectrometry (LCMS); more preferably, the detection method is triple tandem quadrupole mass spectrometry (QQQMS).

[0059] The sample is not particularly limited and can be a biological sample, an environmental sample, a product sample, etc. In a preferred embodiment, the sample is a biological sample; more preferably, the sample is a blood sample. In another preferred embodiment, the sample is a mammalian blood sample; wherein the mammal is a human or other non-human mammal (e.g., a rat blood sample or a beagle blood sample).

[0060] The method of the present invention can also be used to analyze the half-life of polypeptides in blood samples. Specifically, the method comprises the steps of:

[0061] (S1) providing a blood sample;

[0062] (S2) mixing the polypeptide with the blood sample, so that the polypeptide reacts with plasma albumin in the blood sample;

[0063] (S3) enzymatically hydrolyzing the sample using a protease to obtain an enzymatically hydrolyzed sample containing characteristic peptides; and

[0064] (S4) detecting the content of the characteristic peptide, and calculating the concentration of the polypeptide in the blood sample according to the content of the characteristic peptide, and calculating the half-life of the polypeptide according to the concentration;

[0065] Wherein, the polypeptide is a polypeptide that can be coupled to albumin and enzymatically hydrolyzed to form a characteristic peptide; and the sequence of the characteristic peptide is an amino acid sequence having 85% homology with the sequence shown in SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 1 is: NEQELL.

[0066] In a preferred embodiment, in the method, the step (S4) further includes: continuously detecting the content of the characteristic peptide, and then drawing a change curve according to the change of the polypeptide concentration detected at different times, so as to obtain the drug-time curve of the characteristic peptide, and calculating the half-life of the polypeptide according to the drug-time curve.

[0067] In another preferred embodiment, in the method, the reaction time of step (S2) is 4-10 minutes.

[0068] The main advantages of the present invention include:

[0069] (a) The polypeptide of the present invention is coupled to albumin and does not exist in a free state. This method has a stronger specificity than the ELISA method. After research and data analysis, it was determined that the characteristic peptide segment originated from the enzymatic hydrolysis of the conjugate. No such characteristic peptide segment was detected in blank plasma and blank plasma after the same enzymatic hydrolysis treatment. Therefore, the inherent components of plasma will not interfere with the test results; and the method can accurately detect the blood concentration of Abovirta, overcoming the low detection sensitivity of Q-TOF in the prior art, and the limited detection sensitivity of ELISA, MSD and other methods, which makes it difficult to detect the low blood concentration of the polypeptide conjugate at a long time point after administration in the pharmacokinetic (PK) test, so that the half-life of Abovirta can be accurately determined.

[0070] (b) High sensitivity: This method uses the classic MRM mode for data acquisition, which can remove various interferences to the greatest extent. Its detection LLOQ (Lower Limit of Quantification) can reach 1 ng / ml (based on the blood concentration of Abovirta), which is thousands of times more sensitive than the competitive ELISA method or MSD method (electrochemiluminescence detection technology), greatly improving the detection sensitivity.

[0071] In this way, even at low dosages, reliable data on the half-life of the drug can be obtained. Because the 29-34 peptide segment is a characteristic peptide of the compound, the mass spectrometry detection process is not interfered by other proteins or peptides in the blood, and can objectively reflect the changes in the blood drug concentration of the compound.

[0072] (c) Wide quantitative range: This method can use a double calibration curve to extend the quantitative range to 1 ng / ml to 20 μg / ml, effectively covering the blood drug concentration range for intravenous and subcutaneous injections; each calibration curve has at least 6 concentration levels, and the correlation coefficient is greater than 0.99.

[0073] (d) High accuracy: The recovery rate of this method can reach 100% ± 15% from 1 ng / ml to 20 μg / ml, meeting the requirements of the Chinese Pharmacopoeia 2020 ChP <9012> and the requirements of the EMA Guideline on bioanalytical method validation.

[0074] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0075] Example 1 Competitive ELISA method to detect ABT blood concentration experiment

[0076] Package board

[0077] Add 1.5 μg / well ABT antibody to the wells of a 96-well blank ELISA plate, affix the sealing film, and incubate at 2-8°C overnight (16 -23h).

[0078] Washing and blocking

[0079] Wash each well of the ELISA plate 4 times with 300 μL of washing buffer, then pat dry on absorbent paper. Add 200 μL of blocking buffer to the microwells. After applying the sealing film, place the plate on a microplate shaker at room temperature at 500 rpm / min and incubate for 2.5 hours.

[0080] Sample dilution and incubation

[0081] ABT was prepared with whole plasma at 1 mg / mL, incubated at 37°C for 1 hour, and then diluted. Then, the diluted sample was mixed with ABT-HRP working solution at a ratio of 1:1 (ABT-HRP 1:350 dilution). The diluted sample was added to the ELISA plate at 100 μl / well, and the sealing film was attached to the plate. Incubated at room temperature and 500 rpm / min for 2 hours ± 10 minutes. After incubation, the plate was washed 4 times.

[0082] Color

[0083] TMB working solution was added to the ELISA plate at 100 μL / well and incubated at room temperature for 30 min.

[0084] termination

[0085] Add 100 μL / well of the stop solution to the ELISA plate. Within 15 minutes after the stop solution is added, use an ELISA reader to read the OD value at 450 / 630 nm.

[0086] The absorbance measured in the sample concentration range of 100-20000 ng is shown in Table 1.

[0087] Table 1. Absorbance measured in the sample concentration range of 100-20000ng

[0088]

[0089]

[0090] The absorbance measured at 100-20000ng was used to draw a standard curve, as shown in the attached Figure 1 as shown in .

[0091] in conclusion:

[0092] 1. The recovery rate (Recovery%) of each concentration point of the standard (200-20000ng / ml) is between 95.29%-119.75%, and the coefficient of variation (CV value) is between 0.00%-5.44%, which meets the recovery rate acceptance range of 80%-120% and the CV value acceptable range of ±15%.

[0093] 2. The recoveries of the three quality control products (1μg, 3μg, 15μg) were 102.7%, 101.93%, and 108.17%, respectively, and the CV values ​​were 0%, 2.14%, and 0%, respectively, all meeting the recovery rate acceptance range of 80%-120% and the CV value acceptable range of ±15%.

[0094] In summary, this competitive ELISA method can be used to detect ABT blood concentrations within 200-20000 ng / ml.

[0095] Example 2 Experimental steps for detecting ABT blood concentration by QQQMS method

[0096] Simulation sample preparation:

[0097] ABT was added to commercially available Beagle dog plasma to prepare a series of simulated standard plasma solutions ranging from 200 ng / ml to 20 μg / ml (sample concentrations were based on intact ABT molecules). 50 μl of each solution was incubated at 37°C for 5 min, and then 0.2 μg / μl of pancreatic enzyme solution was added at a volume ratio of 1:1. After incubation at 37°C for 4 hours, 50 μl of 0.1% TFA aq. was added to terminate the enzyme cleavage reaction to obtain enzyme cleavage samples containing a characteristic peptide (SEQ ID NO: 1, NEQELL), which were stored in a refrigerator at 2-8°C.

[0098] Sample testing:

[0099] After the enzyme-cut samples were taken out and re-thawed at room temperature, they were treated with solid-phase extraction (SPE) to remove excess impurities such as proteins and lipids in the plasma. The sample concentration was tested in MRM mode of QQQMS, and the calibration curve was fitted to the prepared concentration using the characteristic ion pair peak area. The sample concentration was calculated using the external standard method.

[0100] Detection ion: +1 ion m / z 744→727 (29→34 peptide segment), quantitative limit: 200 ng / ml.

[0101] The simulated test data of ABT blood concentration are shown in Table 2.

[0102] Table 2 ABT blood concentration simulation test data

[0103]

[0104] Conclusion: The MRM mode of QQQMS was used to determine the blood drug concentration of 200-20000ng / ml ABT. The recovery rate of the detection concentration values ​​at each point of the standard curve was between 88.7% and 118.2%; the recovery rate of the detection concentration values ​​of the four quality control products was between 90.0% and 114.7%. All test results were within the acceptable range (recovery rate 80%-120%), meeting the regulatory requirements for biological sample testing, and 200ng / ml was accurate in quantification.

[0105] In the above two embodiments, the comparison of the accuracy of ABT blood concentration detection by competitive ELISA and MRM mode of QQQMS is shown in Table 3:

[0106] Table 3 Comparison of ELISA and QQQMS results

[0107]

[0108] Results: The recovery rates of ABT quality control products (1μg / ml, 3μg / ml, 15μg / ml) in MRM mode of QQQMS were 90%-101.33%, and those in competitive ELISA were 101.93%-108.17%, both of which met the acceptable recovery range of 80%-120% and the regulatory requirements for biological sample testing.

[0109] For ABT quality control products (1μg / ml, 3μg / ml, 15μg / ml), the coefficients of variation between the results detected by the MRM mode of QQQMS and the competitive ELISA method were 2.17%, 8.79%, and 4.62%, respectively, which met the acceptable range of the CV% value of ±15% and met the regulatory requirements for biological sample testing.

[0110] Example 3. Study on the accuracy of the QQQMS method for analyzing the blood concentration of ABT

[0111] According to the detection method of Example 2 above, blood samples of ABT with different concentrations were taken. The specific detection concentrations were 1, 5, 10, 25, 50, 100, 150, and 200 ng / ml, respectively, and the detection was carried out by QQQMS method and ELISA method.

[0112] From the test results, it can be seen that the recovery rate of the MRM mode of QQQMS for the blood drug concentration detection value of ABT is between 91.22% and 111.25%, which meets the acceptable range of recovery rate (% Recovery) of 80%-120%; the sensitivity can reach 1ng / ml, which meets the requirements of the Chinese Pharmacopoeia 2020 ChP <9012> and the requirements of the EMA Guideline on bioanalytical method validation.

[0113] The recovery rate of ABT blood concentration detected by ELSIA method is between 101.25% and 105.81%, which meets the acceptable range of 85%-115% recovery rate, and the sensitivity can reach 50μg / ml, which meets the requirements of ChP 2020 <9012> and EMA Guideline on bioanalytical method validation requirements

[0114] Therefore, the sensitivity of the QQQMS method for detecting ABT blood concentration is much higher than that of the ELSIA method, which can be up to thousands of times higher.

[0115] Conclusion: The detection results of ABT blood peptide concentration by QQQMS in MRM mode were highly consistent with those by traditional competitive ELISA, and the results were reliable. The MRM mode of QQQMS can be used to detect the blood concentration of ABT.

[0116] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for detecting the content of Abovirtide (ABT) in a sample non-diagnostic and non-therapeutic, characterized in that: The following steps are involved: (Z1) providing a plasma sample containing the ABT; (Z2) using a protease to enzymatically hydrolyze the sample to obtain a sample containing characteristic peptides; and (Z3) detecting the content of the characteristic peptide, and calculating the concentration of ABT in the sample according to the content of the characteristic peptide; Wherein, the abovirtide is a polypeptide that can be enzymatically hydrolyzed to form a characteristic peptide, and the protease is a protease that can enzymatically hydrolyze abovirtide to obtain a characteristic peptide.

2. The method according to claim 2, characterized in that The characteristic peptide has an amino acid sequence with more than 85% homology to the sequence shown in SEQ ID NO:

1.

3. The method according to claim 2, characterized in that The amino acid sequence of ibovavirtide is as shown in SEQ ID NO: 2; preferably, position 13 of SEQ ID NO: 2 is modified with an N-{2-[2-(N-(3-maleimidopropionyl)amino)ethoxy]ethoxy}acetyl group.

4. The method according to any one of claims 1 to 3, characterized in that The protease is trypsin; preferably, the sample is selected from the following group: biological samples, environmental samples, product sampling; preferably, the sample is a biological sample; more preferably, the sample is a blood sample.

5. The method according to any one of claims 1 to 4, characterized in that In step (Z3), the content of the hydrolyzed characteristic peptide is detected by liquid chromatography-mass spectrometry (LCMS); preferably, the liquid chromatography-mass spectrometry (LCMS) is a mass spectrometry method selected from the following group: triple tandem quadrupole mass spectrometry (QQQMS), tandem quadrupole composite linear ion trap mass spectrometry (Q-TRAP), quadrupole time-of-flight mass spectrometry (Q-TOF) and ion trap-time-of-flight mass spectrometry (IT-TOF).

6. A method for non-diagnostic and non-therapeutic analysis of the half-life of elbovirtide in a blood sample, characterized in that The method comprises the following steps: (S1) providing a blood sample; (S2) mixing the ibovavirtide with the blood sample, so that the ibovavirtide reacts with the plasma albumin in the blood sample, preferably for 5-60 minutes; (S3) enzymatically hydrolyzing the sample using a protease to obtain a sample containing enzymatically hydrolyzed characteristic peptides; and (S4) detecting the content of the enzymatically hydrolyzed characteristic peptide, calculating the concentration of ibovavir in the blood sample based on the content of the characteristic peptide, and calculating the half-life of ibovavir based on the concentration; Wherein, the ibovavirtide is a polypeptide that can be enzymatically hydrolyzed to form a characteristic peptide, and the protease is a protease that can enzymatically hydrolyze ibovavirtide to obtain a characteristic peptide; preferably, the characteristic peptide has an amino acid sequence that is more than 85% homologous to the sequence shown in SEQ ID NO:

1.

7. The method according to claim 6, characterized in that The amino acid sequence of ibovavirtide is as shown in SEQ ID NO: 2; preferably, position 13 of SEQ ID NO: 2 is modified with an N-{2-[2-(N-(3-maleimidopropionyl)amino)ethoxy]ethoxy}acetyl group.

8. The method according to claim 6 or 7, characterized in that In the method, the step (S4) also includes: continuously detecting the content of the enzymatic characteristic peptide, and then drawing a change curve according to the change of the concentration of the ibovavirtide detected at different times, so as to obtain the drug-time curve of the polypeptide, and calculating the half-life of the ibovavirtide according to the drug-time curve.

9. The method according to any one of claims 6 to 8, characterized in that In step (S4), the content of the enzymatic characteristic peptide is measured by liquid chromatography-mass spectrometry (LCMS); preferably, the liquid chromatography-mass spectrometry (LCMS) is a mass spectrometry method selected from the following group: triple tandem quadrupole mass spectrometry (QQQMS), tandem quadrupole composite linear ion trap mass spectrometry (Q-TRAP), quadrupole time-of-flight mass spectrometry (Q-TOF) and ion trap-time-of-flight mass spectrometry (IT-TOF).

10. A kit, characterized in that: The kit comprises a characteristic peptide having an amino acid sequence with 85% homology to the sequence shown in SEQ ID NO:1.

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