Method for detecting polypeptide drugs in biological sample by LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention relates to a method for detecting polypeptide drugs in a biological sample by LC-MS / MS. The method comprises the following steps: adding a precipitator into the biological sample, carrying out centrifugal vortex mixing uniformly, and taking supernate; carrying out reversed-phase high-performance liquid chromatography separation on the supernate by adopting an octadecylsilane chromatographic column, carrying out tandem mass spectrometry detection analysis, scanning to obtain a sample spectrogram, and substituting the sample spectrogram into a standard curve to calculate the concentration of the polypeptide drug. According to the detection and analysis method, sample treatment is simple and easy to operate, the recovery rate is high, and polypeptide drugs in non-clinical biological samples can be rapidly and accurately determined.

Description

Technical Field

[0001] The invention belongs to the technical field of biological sample drug analysis, and specifically relates to a method for detecting polypeptide drugs in biological samples by LC-MS / MS. Background Art

[0002] Experts at home and abroad generally believe that the general direction of the new coronavirus mutation is to be less pathogenic, more prone to upper respiratory tract infection, and have a shorter incubation period. Studies have shown that peptide drugs can competitively block the fusion of SARS-CoV-2 virus into cells, reduce the risk of viral infection, and prevent further spread of the virus. Peptide drugs are administered by inhalation and are mainly distributed in the lungs and throat tissues, with low drug concentrations in plasma. Therefore, the establishment of analytical methods for the determination of peptide drugs in biological matrices faces many challenges.

[0003] Enzyme-linked immunosorbent assay (ELISA) and radioisotope labeling are the most commonly used methods for quantitative detection of peptide drugs. In recent years, liquid chromatography-mass spectrometry (LC-MS / MS) technology has developed rapidly. Its high specificity, high sensitivity, wide application, simplicity and rapidity make it an important means for quantitative analysis of peptide drugs in vivo.

[0004] In the process of LC-MS / MS detection of peptide drugs, the matrix components of biological samples will interfere with the ionization of the target during the mass spectrometry ionization process (ion enhancement or ion suppression), resulting in an increase or decrease in the detection signal, which in turn leads to deviations in the detection results. Therefore, sample pretreatment is the most critical and time-consuming step in LC-MS / MS detection of biological samples. Therefore, providing a simple, easy-to-operate, high-recovery, and short-time sample pretreatment method has become a technical problem that needs to be solved in LC-MS / MS detection of the content of peptide drugs in biological samples. Summary of the invention

[0005] The purpose of the present invention is to provide a LC-MS / MS method for detecting polypeptide drugs in biological samples which is simple to operate and has a high recovery rate.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for detecting polypeptide drugs in biological samples by LC-MS / MS, the method comprising the following steps:

[0008] S1. Biological sample pretreatment: add precipitant to the biological sample, vortex mix, centrifuge, and take the supernatant;

[0009] S2. Detection of polypeptide drugs: The supernatant in S1 is separated by reverse phase high performance liquid chromatography using an octadecylsilane column, and analyzed by tandem mass spectrometry to obtain a spectrum of polypeptide drugs;

[0010] S3. Calculate the concentration of peptide drugs: perform linear least squares regression calculation on the theoretical concentration of the peptide compound in the standard curve based on the ratio of the peak area of ​​the peptide drug in the biological sample to the peak area of ​​the internal standard, and calculate the concentration of the peptide drug in the biological sample using the obtained regression equation.

[0011] In some embodiments of the present invention, the biological sample is plasma or a tissue sample. Preferably, the tissue sample is a lung tissue sample.

[0012] In some embodiments of the present invention, the precipitant is a protic organic solvent or a protic organic solvent containing an organic acid.

[0013] In some embodiments of the present invention, the protic organic solvent is methanol, ethanol, isopropanol. Preferably, the protic organic solvent is methanol.

[0014] In some embodiments of the present invention, the organic acid in the protic organic solvent containing an organic acid is formic acid, acetic acid, trifluoroformic acid, or trifluoroacetic acid. Preferably, the organic acid is formic acid.

[0015] In some embodiments of the present invention, the concentration of the organic acid in the protic organic solvent containing the organic acid is 0.05%-2%. Preferably, the concentration of the organic acid is 1%.

[0016] In some embodiments of the present invention, the protic organic solvent containing an organic acid is methanol containing 1% formic acid.

[0017] In some embodiments of the present invention, the volume ratio of the biological sample to the precipitant is 1:2-6. Preferably, the volume ratio of the biological sample to the precipitant is 1:3, 1:4 or 1:6.

[0018] In some embodiments of the present invention, when the biological sample is plasma, a precipitant is added, the mixture is vortexed and then centrifuged to obtain a supernatant.

[0019] In some embodiments of the present invention, when the biological sample is a tissue sample, a precipitant is added, vortexed and mixed, and then centrifuged, and the supernatant is taken and vortexed again and mixed, and then centrifuged to take the supernatant.

[0020] In some embodiments of the present invention, the polypeptide drug has the following general structure:

[0021] Formula I:

[0022] R1-Z1-DISGINASVVNIQ-X1-EI-X2-X3-L-X4-X5-VAK-X6-LN-X7-SLIDLQEL-Z2-R2,

[0023] Or formula II: R1-Z1-QYGSFCTQLNRALTGIAVEQ-Z2-R2;

[0024] Or formula III: R1-Z1-YQDVNCTEVPVAIHADQLTP-Z2-R2;

[0025] Wherein, X1, X2, X3, X4, X5, X6 and X7 are each independently selected from one of D-type or L-type amino acids; Z1 and Z2 do not exist, or Z1 and Z2 are each independently selected from one or more D-type or L-type amino acids; the D-type or L-type amino acids are selected from glutamic acid (E), aspartic acid (D), lysine (K), asparagine (N), arginine (R), glutamine (Q), histidine (H) and phenylalanine (F);

[0026] R1 is an N-terminal modification group, which is absent or is an amino protecting group connected to the primary amine functional group of the N-terminal amino acid;

[0027] R2 is a C-terminal modification group, which is absent or is a polypeptide molecule of a carboxyl protecting group connected to the carboxyl functional group of the C-terminal amino acid.

[0028] Preferably, in the general formula I, X1, X2, X3, X4, X5, X6 and X7 are lysine (K), aspartic acid (D), arginine (R), asparagine (N), glutamic acid (E), asparagine (N) and glutamic acid (E), respectively;

[0029] Preferably, X1, X2, X3, X4, X5, X6 and X7 are glutamic acid (E), aspartic acid (D), arginine (R), glutamic acid (E), glutamic acid (E), asparagine (N) and glutamic acid (E), respectively;

[0030] Preferably, X1, X2, X3, X4, X5, X6 and X7 are glutamic acid (E), lysine (K), arginine (R), glutamic acid (E), glutamic acid (E), glutamic acid (E) and lysine (K), respectively.

[0031] More preferably, the polypeptide drug is selected from the following polypeptides:

[0032] ACV01: DISGI NASVV NIQKE IDRLN EVAKN LNESL IDLQE L, as shown in SEQ ID NO. 1;

[0033] ACV02: DISGI NASVV NIQEE IDRLE EVAKN LNESL IDLQE L, as shown in SEQ ID NO. 2;

[0034] ACV03: DISGI NASVV NIQEE IKRLE EVAKE LNKSL IDLQE L, as shown in SEQ ID NO. 3;

[0035] Most preferably, the polypeptide drug is a polypeptide with the following structure:

[0036] ACV03: DISGI NASVV NIQEE IKRLE EVAKE LNKSL IDLQE L, as shown in SEQ ID NO.3, the N-terminus of the polypeptide molecule is free or connected to an amino protecting group, and the C-terminus of the polypeptide molecule is free or connected to a carboxyl protecting group.

[0037] Preferably, the polypeptide drug structure described in Formula II is ACV04: QYGSF CTQLN RALTG IAVEQ, as shown in SEQ ID NO.4;

[0038] Preferably, the polypeptide drug of general formula (III) is ACV05: YQDVN CTEVP VAIHA DQLTP, as shown in SEQ ID NO.5;

[0039] In some embodiments of the present invention, the reverse phase high performance liquid chromatography separation conditions are as follows:

[0040] Octadecylsilane column: Waters BEH C18 2.1*50mm, 1.7μm column; mobile phase A is an aqueous solution containing 0.3% formic acid, mobile phase B is acetonitrile containing 0.3% formic acid, column temperature is 40°C, flow rate is 0.4ml / min, and elution gradient conditions are as follows:

[0041] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 0.5 80 20 2.0 10 90 2.9 10 90 3.0 80 20 4.0 80 20

[0042] In some embodiments of the present invention, the tandem mass spectrometry detection analysis conditions are as follows:

[0043] Ionization mode: ESI+; spray voltage (IS): 5500KV; ion source temperature (TEM): 550℃; nebulizer gas (GAS1): 60psi; auxiliary nebulizer gas (GAS2): 60psi; curtain gas (CUR): 20psi; collision gas (CAD): 10psi; scanning mode: multiple reaction monitoring (MRM).

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The LC-MS-MS detection method of the present invention improves the detection sensitivity of polypeptide drugs in biological matrices, solves the problems of matrix interference and poor recovery rate, and has a simple operation method, and can be used for rapid quantitative detection of polypeptide compounds in non-clinical biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the MRM chromatogram of the sample obtained after plasma was extracted with tert-butyl methyl ether in Comparative Example 1 (A is ACV03; B is bivalirudin);

[0047] Figure 2 This is the MRM chromatogram of the sample obtained after pretreatment with tert-butyl methyl ether-ethyl acetate (1:1) in Comparative Example 1 (A is ACV03; B is bivalirudin);

[0048] Figure 3 Example 1 MRM chromatogram of double blank plasma samples (A is ACV03; B is bivalirudin);

[0049] Figure 4 This is the MRM chromatogram of the blank plasma sample of Example 1 (A is ACV03; B is bivalirudin);

[0050] Figure 5 The MRM chromatogram of the plasma quantitative lower limit sample of Example 1 (A is ACV03; B is bivalirudin);

[0051] Figure 6 The MRM chromatogram of the upper limit sample of plasma quantification in Example 1 (A is ACV03; B is bivalirudin);

[0052] Figure 7 This is a typical standard curve diagram of the polypeptide compound in plasma in Example 1;

[0053] Figure 8 This is the MRM chromatogram of high concentration quality control in mouse lung tissue in Example 2 (A is ACV03; B is bivalirudin);

[0054] Fig. 9 This is the MRM chromatogram of the high-concentration matrix-containing sample in the high-concentration mouse lung tissue in Example 2 (A is ACV03; B is bivalirudin);

[0055] Fig.10 This is the MRM chromatogram of the quantitative lower limit sample of mouse lung tissue in Example 2 (A is ACV03; B is bivalirudin);

[0056] Fig.11 This is the MRM chromatogram of the upper limit sample of mouse lung tissue quantification in Example 2 (A is ACV03; B is bivalirudin);

[0057] Fig.12 This is the MRM chromatogram of the high-concentration quality control sample of mouse lung tissue in Example 2 (A is ACV03; B is bivalirudin);

[0058] Fig.13 This is the MRM chromatogram of the low-concentration quality control sample of mouse lung tissue in Example 2 (A is ACV03; B is bivalirudin);

[0059] Fig.14 This is a typical standard curve diagram of the polypeptide compounds in the mouse lung tissue sample in Example 2. DETAILED DESCRIPTION

[0060] The present invention will be further described below in conjunction with examples, which are intended only to illustrate the present invention rather than to limit the present invention.

[0061] Example 1: Method for detecting polypeptide compounds in plasma by LC-MS / MS

[0062] 1. Materials and instruments

[0063] High performance liquid chromatography-tandem mass spectrometry was used, equipped with an electrospray ionization (ESI) source (TRIPLE QUAD 4500MD, AB SCIEX, USA), the liquid chromatography separation mode was reversed phase chromatography, and the detector was a triple quadrupole tandem mass spectrometer.

[0064] Chromatographic column: Waters BEH C18 2.1*50mm, 1.7μm.

[0065] Peptide compound: ACV03

[0066] 2. Experimental plan

[0067] 2.1. Preparation of calibration standard working solution and quality control working solution

[0068] Weigh the reference polypeptide compound ACV03, add an appropriate volume of 80% methanol-water solution, adjust the pH to about 8.5 with 1 mg / ml sodium hydroxide solution, vortex thoroughly until completely dissolved, add an appropriate amount of 80% methanol to volume and mix well to prepare a 1 mg / ml polypeptide compound solution. Take the 1 mg / ml polypeptide compound solution and use 80% methanol-water solution to prepare calibration standard working solutions with concentrations of 100, 200, 400, 1000, 2000, 4000, 10000, and 20000 ng / ml and quality control working solutions with concentrations of 300, 600, 6000, and 16000 ng / ml.

[0069] 2.2 Preparation of internal standard working solution

[0070] 1 mg of bivalirudin was dissolved in dimethyl sulfoxide to prepare an internal standard stock solution with a concentration of 1.00 mg / ml. The internal standard stock solution with a concentration of 1.00 mg / ml was dissolved in methanol to prepare an internal standard working solution with a concentration of 100 ng / ml.

[0071] 2.3 Preparation of test samples

[0072] 2.3.1. Preparation of calibration standards

[0073] Take 5 μl of each calibration standard working solution with a concentration of 100, 200, 400, 1000, 2000, 4000, 10000, and 20000 ng / ml in 2.1, add 95 μl of blank plasma, mix evenly, and obtain calibration standard samples with a concentration of 5, 10, 20, 50, 100, 200, 500, and 1000 ng / ml.

[0074] 2.3.2 Preparation of quality control samples

[0075] Take 5 μl of each of the quality control working solutions with concentrations of 300, 600, 6000, and 16000 ng / ml in 2.1, add 95 μl of blank plasma, mix well, and obtain quality control samples with concentrations of 15, 30, 600, and 800 ng / ml.

[0076] 2.3.3. Plasma sample pretreatment

[0077] Take 100 μl of the sample prepared under 2.3.1 and 2.3.2, add 400 μl of methanol precipitant containing 100 ng / ml internal standard working solution (bivalirudin), mix well, centrifuge at 13000 rpm and 4°C for 10 min, and transfer the supernatant to the sample injection plate.

[0078] 2.3.4 Preparation of unextracted quality control samples

[0079] Take 100 μl of blank plasma and add 400 μl of methanol precipitation, and repeat 10 times. Take the supernatant and combine it to obtain blank supernatant. Take 95 μl of blank supernatant and add 5 μl of quality control working solution with concentrations of 300, 600, 6000, and 16000 ng / ml, and then add 400 μl of 100 ng / ml internal standard working solution prepared from blank supernatant. Mix well and transfer to the injection plate to obtain unextracted quality control samples with concentrations of 15, 30, 600, and 800 ng / ml.

[0080] 2.3.5 Preparation of matrix-free quality control samples

[0081] Take 95 μl of methanol, add 5 μl of quality control working solution with concentrations of 300, 600, 6000, and 16000 ng / ml and 400 μl of internal standard containing 100 ng / ml, mix well, and transfer to the sample injection plate to obtain matrix-free quality control samples with concentrations of 15, 30, 600, and 800 ng / ml.

[0082] 2.3.6. Preparation of selective samples

[0083] Double blank sample: Take 100 μl of blank plasma without adding standard working solution and internal standard, mix well, centrifuge at 13000 rpm and 4°C for 10 min, and transfer the supernatant to the sample injection plate.

[0084] Blank sample: Take 95 μl of blank plasma, add 5 μl of internal standard, do not add standard working solution, mix well, centrifuge at 13000 rpm and 4°C for 10 min, and transfer the supernatant to the sample injection plate.

[0085] 2.4. Sample testing

[0086] The samples prepared in 2.3 were tested under the following LC-MS / MS conditions:

[0087] HPLC conditions: chromatographic column: Waters BEH C18 2.1*50mm, 1.7μm; mobile phase: 0.3% formic acid aqueous solution (A), 0.3% formic acid acetonitrile (B), column temperature 40°C, injection volume 15μl, autosampler 4°C, flow rate 0.4ml / min, needle wash solution: acetonitrile-isopropanol (1:1), elution gradient is as follows:

[0088] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 0.5 80 20 2.0 10 90 2.9 10 90 3.0 80 20 4.0 80 20

[0089] Mass spectrometry conditions: ionization mode: ESI+; spray voltage (IS): 5500KV; ion source temperature (TEM): 550°C; nebulizer gas (GAS1): 60psi; auxiliary nebulizer gas (GAS2): 60psi; curtain gas (CUR): 20psi; collision gas (CAD): 10psi; scanning mode: multiple reaction monitoring (MRM); the qualitative and quantitative ion pairs, dwell time, collision energy and other parameters of peptide drugs and their internal standards are as follows:

[0090]

[0091] 3. Experimental results

[0092] 3.1 Recovery rate

[0093] The results of the recovery of peptide compounds in plasma are shown in Table 1.

[0094] Table 1 Recovery rate of peptide compounds in plasma* (each sample was repeated 6 times)

[0095]

[0096]

[0097] *Recovery rate calculation method: The ratio of the peak area of ​​the target ion chromatogram peak to the peak area of ​​the internal standard ion chromatogram peak in the sample chromatogram is used as the response, and a linear regression equation is made with the corresponding concentrations.

[0098] The experimental results in Table 1 show that the recovery rates of the polypeptide compounds in plasma ranged from 92.2% to 101.8%, with an overall CV% of 4.2%, indicating a good recovery rate.

[0099] 3.2 Matrix Effects

[0100] The results of the matrix effect of peptide compounds in plasma are shown in Table 2.

[0101] Table 2 Matrix effects of peptide compounds in plasma*

[0102]

[0103] *Matrix effects were calculated using the chromatographic peak area responses of the peptide drug and internal standard.

[0104] The results in Table 2 show that the matrix effect of plasma samples was 71.7% to 87.7% when the supernatant test solution obtained by extracting plasma samples in 2.3.2 was used for testing, and the CV% at each concentration level was no more than 11.3%, indicating a good matrix effect.

[0105] 3.3. Validation of bioanalytical methodology for detection of peptide drugs in plasma by LCMS-MS

[0106] 3.3.1 Selectivity

[0107] The selective samples were tested according to the sample testing conditions in Example 1, and the results were as follows: Figure 3 , Figure 4 The results showed that there were no obvious peaks of internal standard bivalirudin and ACV03 peptide drugs in the double blank and blank, and the blank plasma matrix and internal standard did not affect the detection of peptide drugs.

[0108] 3.3.2 Sensitivity and linear relationship

[0109] The sensitivity and linear relationship were investigated using the calibration standard samples with concentrations of 5, 10, 20, 50, 100, 200, 500, and 1000 ng / ml obtained in 2.3.1. The lower limit of quantification of polypeptide compounds in plasma (5 ng / ml) is Figure 5 As shown, Figure 5A in the middle is the peptide drug ACV03, with a peak area of ​​770. Figure 6 B is the internal standard bivalirudin, with a peak area of ​​729000. The upper limit of quantification of peptide compounds in plasma (1000ng / ml) is Figure 6 As shown, Figure 6 A in the middle is the peptide drug ACV03, with a peak area of ​​193,000. Figure 6 In the figure, B is the internal standard bivalirudin, with a peak area of ​​740,000.

[0110] The back-calculated concentration results of the calibration standards in plasma are shown in Table 3.

[0111] Table 3 Back-calculated concentrations of calibration standards in plasma

[0112]

[0113] According to the linear regression equation: y = 0.251x-0.000234, the typical standard curve of peptide compound drugs in plasma was obtained. Figure 7 , and its correlation coefficient R = 0.9924, confirming that the standard curve has a good linear relationship, and the method detection limit is determined to be 5ng / ml. It can be seen that the detection method in Implementation 1 has good sensitivity and linearity in detecting peptide drugs in plasma.

[0114] Example 2: LC-MS / MS Bioanalysis Method for Detecting Peptide Drugs in Mouse Lung Tissue

[0115] 1. Experimental plan

[0116] 1.1. Preparation of test samples

[0117] 1.1.1 Preparation of homogenate

[0118] Methanol homogenate containing 1% formic acid was added to the blank lung tissue of mice, and the volume ratio of the blank lung tissue to the homogenate was 1:6. After homogenization, a blank homogenate was obtained.

[0119] 1.1.2 Preparation of calibration standard samples and quality control samples

[0120] Take 95 μl of the above mouse lung tissue homogenate, add 5 μl of calibration standard working solution with concentrations of 5, 10, 20, 50, 100, 200, 500, 1000 ng / ml and 5 μl of quality control working solution with concentrations of 300, 600, 6000, 16000 ng / ml, respectively, to obtain calibration standard and quality control samples. Add 400 μl of 100 ng / ml internal standard working solution, mix well, centrifuge at 13000 rpm for 10 min, take the supernatant to a 96-well plate, centrifuge at 4000 rpm for 10 min, and finally take the supernatant and transfer it to a new treatment plate.

[0121] 1.1.3 Preparation of Matrix-Containing Samples

[0122] Take a mouse lung tissue homogenate sample, add 500 μl of methanol, mix well, remove 100 μl of the supernatant after centrifugation, blow dry with nitrogen, add 5 μl of quality control working solution, 400 μl of internal standard working solution, and 100 μl of methanol to obtain a sample containing matrix.

[0123] 1.1.4 Preparation of Matrix-free Samples

[0124] Take 5 μl of quality control working solution, add 95 μl of methanol, 400 μl of internal standard working solution, and 100 μl of methanol, mix well to obtain a matrix-free sample.

[0125] 1.2 Detection Method

[0126] The LC-MS / MS detection conditions in Example 1 were used for detection.

[0127] 1.3 Experimental Results

[0128] 1.3.1. Back-calculated concentration of calibration standards in tissues

[0129] Table 4 Back-calculated concentrations of calibration standards in tissues

[0130]

[0131] *Indicates outliers, which are not included in the calculation of the mean.

[0132] 1.3.2 Accuracy of quality control samples in tissues

[0133] Table 5 Accuracy of quality control samples in tissues

[0134]

[0135] *Six samples were processed in parallel for each concentration, excluding outliers within the batch, i.e. n = 4, indicating that the mean was calculated based on 4 measured concentrations.

[0136] 1.3.3 Sensitivity and linear relationship

[0137] Detect the lower calibration standard sample in 1.1.2 of Example 2 to obtain the lower limit sample of mouse lung tissue quantification Fig.10 , and the upper limit of quantitation sample Fig.11 .That Fig.10 A is the peptide drug detection peak with a peak area of ​​818, and B is the internal standard detection peak with a peak area of ​​738000; Fig.11 In the figure, A is the peptide drug detection peak with a peak area of ​​247,000; B is the internal standard detection peak with a peak area of ​​803,000.

[0138] The above test sample results show that: the chromatographic separation effect of polypeptide compounds at low and high concentration levels is good, with good sensitivity and accuracy; under the previous processing and analysis methods, the linear regression equation of the polypeptide compound is: y=0.315x-0.000433; the accuracy ranges of the calibration standards and quality control samples are -10.4%~14.0% and -1.8%~8.1%, respectively, see Tables 4 and 5 for details.

[0139] Therefore, the peptide compounds in lung tissue were well extracted and separated under this pretreatment method, the chromatographic peaks were well separated by LC-MS / MS, and the accuracy of calibration standards and quality control samples met the acceptance criteria.

[0140] Comparative Example 1: Treatment of mouse plasma by liquid-liquid extraction pretreatment method

[0141] Take 50 μl of each calibration standard working solution and quality control working solution of each concentration in Example 1, and add 1 ml of tert-butyl methyl ether and 50% tert-butyl methyl ether-50% ethyl acetate in 15 ml centrifuge tubes respectively, and then add 50 μl of 100 ng / ml internal standard working solution, mix well, centrifuge at 4000 rpm for 10 min, take the supernatant, blow dry with nitrogen, and finally re-dissolve with 200 μl of 50% methanol-50% ultrapure water (containing 0.3% formic acid). The internal standard working solution was tested simultaneously using the LC-MS / MS conditions in Example 1, and the test results are shown in Figure 1 and Figure 2 .

[0142] like Figure 1 and Figure 2 The test results show that when using tert-butyl methyl ether and 50% tert-butyl methyl ether-50% ethyl acetate solvents for extraction, no polypeptide compounds are retained, indicating that the extraction effects of the above-mentioned pretreatment solvents are not good and cannot meet the detection requirements.

[0143] Comparative Example 2: Solid Phase Extraction Method for Treatment of Mouse Plasma

[0144] 1. Preparation of test samples

[0145] 1.1. Preparation of solid phase extraction (non-acidified) samples

[0146] Take one portion of the calibration standard sample in Example 1, add 20 μl of the internal standard working solution, first use 1 ml × 2 times of methanol on the solid phase extraction column, then add 1 ml × 2 times of 3% NH3·H2O and wait for it to drain, then use a pipette to draw the non-acidified sample to the activated solid phase extraction column, add 1 ml × 2 times of 3% NH3·H2O-H2O, 1 ml × 2 times of 50% methanol-50% acetonitrile (containing 3% NH3·H2O) in sequence; add 1 ml of ultrapure water, and finally add 300 μl or 500 μl of acetonitrile-methanol-water (4:1:1, containing 3% formic acid) for elution, collect all the eluates in a 2 ml centrifuge tube and mix them evenly to obtain a solid phase extracted (non-acidified) sample;

[0147] 1.2. Preparation of solid phase extraction (acidification) samples

[0148] Take one portion of the calibration standard sample in Example 1, add 20 μl of the internal standard working solution, then add 200 μl of 10% H3PO4, mix well, centrifuge at 13000 rpm for 1 min, and obtain an acidified sample for later use. First use 1 ml of methanol twice on the solid phase extraction column, then add 1 ml of water twice; after it is drained, use a pipette to draw the acidified sample to the activated solid phase extraction column, add 1 ml of 3% NH3·H2O-H2O twice, 1 ml of methanol twice, and finally elute with 500 μl of 2% formic acid-methanol, collect all the eluates in a 2 ml centrifuge tube and mix well to obtain a solid phase extraction (acidified) sample

[0149] 1.3. Preparation of untreated solution (unacidified)

[0150] Take 190 μl of methanol, add 10 μl of quality control working solution and 20 μl of standard, mix well, and obtain a matrix-free sample (unacidified).

[0151] 2. Experimental results

[0152] 2.1. The sample was analyzed using the detection conditions of LC-MS / MS in Implementation 1. The test results are shown in Table 6.

[0153] Table 6 Recovery rates of different solid phase extraction pretreatment methods

[0154]

[0155] As shown in Table 6, the peak areas of the peptide compounds and their internal standards in the acidified or non-acidified samples after extraction by solid phase extraction are significantly lower than those in the untreated solution samples, indicating that the recovery rate of the solid phase extraction method is low and cannot meet the detection requirements.

[0156] Comparative Example 3: Treatment of mouse lung tissue with an organic solvent containing no organic acid

[0157] 1. Treating mouse lung tissue with protic organic solvent methanol

[0158] Take the blank lung tissue of mice and add methanol homogenate. The volume ratio of blank lung tissue to homogenate is 1:3. After homogenization, a blank homogenate is obtained. Take 95 μl of the blank homogenate and add 5 μl of the quality control working solution. Mix well and incubate at 13000 rpm. Centrifuge at 4°C for 10 min, take the supernatant to obtain the quality control sample of mouse lung tissue treated with methanol; take 100 μl of blank homogenate, add 500 μl of methanol, mix evenly, take 100 μl of supernatant after centrifugation, blow dry with nitrogen, add 5 μl of quality control working solution, 400 μl of internal standard working solution, and 100 μl of methanol to obtain a matrix-containing sample; take 5 μl of quality control working solution, add 195 μl of methanol, 400 μl of internal standard working solution, and 100 μl of methanol, mix evenly to obtain a matrix-free sample, and use the LCMS / MS conditions of Example 1 for quantitative analysis. The matrix effect after normalization is 89.6% to 101%, and the CV% is 7.13%. Although the matrix effect basically meets the requirements, the peak area of ​​the polypeptide compound in the high concentration 6000 ng / ml matrix-free sample ( Figure 8 A) is significantly lower than Fig. 9 A contains the peak area of ​​peptide compounds in the matrix sample, indicating that the recovery rate of peptide compounds is low when methanol is used as a precipitant to treat the tissue.

[0159] 2. Treating mouse lung tissue using different types of buffer solutions

[0160] Take formic acid, ammonia, 10mM EDTA-K2 and methanol to prepare 2% formic acid-methanol, 2% ammonia-methanol, 50% methanol (containing 10mM EDTA-K2) buffer solutions, and then dilute the mouse lung tissue homogenate at a ratio of 1:2 (tissue and methanol are homogenized at a ratio of 1:3) to obtain 3 kinds of mouse blank lung tissue homogenates under different pH conditions, and obtain mouse blank lung tissue homogenates containing 1% formic acid-methanol, 1% ammonia-methanol, and 70% methanol (containing 5mM EDTA-K2). Take 95μl of the three kinds of mouse blank tissue homogenates of different pH, add 5μl of working solution, mix well, add 400μl of internal standard working solution containing methanol precipitant, mix and centrifuge to obtain supernatant, and obtain tissue samples. The sample detection conditions of Example 1 are adopted, and the results are shown in Table 7.

[0161] Table 7 Peak area values ​​of tissue samples pre-treated with different buffer solutions

[0162]

[0163] The experimental results in Table 7 show that compared with 1% ammonia-methanol and 70% methanol (containing 5mM EDTA-K2), the peak area ratio of peptide drugs to internal standard peak area in the LC-MS / MS detection method was larger when 1% formic acid-methanol was used as the homogenate to treat mouse lung tissue, confirming that after using 1% formic acid-methanol to treat mouse lung tissue, the LC-MS / MS method has a higher response to peptide drug detection.

[0164] 3. Treat mouse lung tissue with formic acid-methanol at different concentration ratios

[0165] Take formic acid solutions of 2% formic acid-methanol, 1% formic acid-methanol, and 0.01% formic acid-methanol, dilute the mouse lung tissue homogenate at a ratio of 1:2 (methanol and tissue are homogenized at a ratio of 1:3), and obtain blank mouse lung tissue samples containing different proportions of formic acid, which are 1% formic acid-methanol, 0.5% formic acid-methanol, and 0.05% formic acid-methanol. Take 95μl of the blank mouse tissue homogenate containing different proportions of formic acid, add 5μl of working solution, mix well, add 400μl of internal standard working solution containing methanol precipitant, mix, and centrifuge to obtain the supernatant to obtain the tissue sample. The sample detection conditions of Example 1 are adopted, and the results are shown in Table 8.

[0166] Table 8 Peak area values ​​of tissue samples pretreated with formic acid buffer solutions at different concentrations

[0167] name Peptide compound peak area Internal standard peak area 1% Formic acid-methanol 145000 592000 0.5% Formic acid-methanol 115 9380 0.05% Formic acid-methanol 65800 374000

[0168] The results in Table 8 show that when the tissue is homogenized with 1% formic acid-methanol homogenate, the ratio of the peak area of ​​the polypeptide compound to the peak area of ​​the internal standard is larger, indicating that the LC-MS / MS detection of polypeptide compounds is more responsive when 1% formic acid-methanol is used for tissue pretreatment.

Claims

1. A method for detecting polypeptide drugs in biological samples by LC-MS / MS, characterized in that: The method comprises the following steps: S1. Biological sample pretreatment: add precipitant to the biological sample, vortex mix, centrifuge, and take the supernatant; S2. Detection of polypeptide drugs: The supernatant in S1 is separated by reverse phase high performance liquid chromatography using an octadecylsilane column, and analyzed by tandem mass spectrometry to obtain a spectrum of polypeptide drugs; S3. Calculate the concentration of peptide drugs: perform linear least squares regression calculation on the theoretical concentration of the peptide compound in the standard curve based on the ratio of the peak area of ​​the peptide drug in the biological sample to the peak area of ​​the internal standard, and calculate the concentration of the peptide drug in the biological sample using the obtained regression equation.

2. The method according to claim 1, characterized in that The biological sample is plasma or a tissue sample; preferably, the tissue sample is a lung tissue sample; and the precipitant is a protic organic solvent or a protic organic solvent containing an organic acid.

3. The method according to claim 2, characterized in that The protic organic solvent is methanol, ethanol, or isopropanol; preferably, the protic organic solvent is methanol.

4. The method according to claim 2, characterized in that: The organic acid in the protic organic solvent containing organic acid is formic acid, acetic acid, trifluoroformic acid, trifluoroacetic acid; preferably, the organic acid is formic acid.

5. The method according to claim 4, characterized in that The organic acid in the protic organic solvent containing organic acid is formic acid, acetic acid, trifluoroformic acid, trifluoroacetic acid. Preferably, the organic acid is formic acid, and the organic acid concentration in the protic organic solvent containing organic acid is 0.05%-2%; preferably, the concentration of the organic acid is 1%, and more preferably, the protic organic solvent is 1% formic acid in methanol.

6. The method according to claim 1, characterized in that The volume ratio of the biological sample to the precipitant is 1:2-6; preferably, the volume ratio of the biological sample to the precipitant is 1:3, 1:4 or 1:

6.

7. The method according to claim 1, characterized in that The polypeptide drug has the following general structure: Formula I: R1-Z1-DISGINASVVNIQ-X1-EI-X2-X3-L-X4-X5-VAK-X6-LN-X7-SLIDLQEL-Z2-R2; Or formula II: R1-Z1-QYGSFCTQLNRALTGIAVEQ-Z2-R2; Or formula III: R1-Z1-YQDVNCTEVPVAIHADQLTP-Z2-R2; Wherein, X1, X2, X3, X4, X5, X6 and X7 are each independently selected from one of D-type or L-type amino acids, Z1 and Z2 do not exist, or Z1 and Z2 are each independently selected from one or more D-type or L-type amino acids; the D-type or L-type amino acids are selected from glutamic acid E, aspartic acid D, lysine K, asparagine N, arginine R, glutamine Q, histidine H and phenylalanine F; R1 is an N-terminal modification group, which is absent or is an amino protecting group connected to the primary amine functional group of the N-terminal amino acid; R2 is a C-terminal modification group, which is absent or is a polypeptide molecule of a carboxyl protecting group connected to the carboxyl functional group of the C-terminal amino acid; Preferably, the polypeptide drug structure described in Formula II is ACV04: QYGSF CTQLN RALTG IAVEQ, as shown in SEQ ID NO.4; Preferably, the polypeptide drug described in general formula III is ACV05: YQDVN CTEVP VAIHA DQLTP, as shown in SEQ ID NO.

5.

8. The method according to claim 7, characterized in that In the general formula I, X1, X2, X3, X4, X5, X6 and X7 are lysine K, aspartic acid D, arginine R, asparagine N, glutamic acid E, asparagine N and glutamic acid E, respectively; or X1, X2, X3, X4, X5, X6 and X7 are glutamic acid E, aspartic acid D, arginine R, glutamic acid E, glutamic acid E, asparagine N and glutamic acid E, respectively; or X1, X2, X3, X4, X5, X6 and X7 are glutamic acid E, lysine K, arginine R, glutamic acid E, glutamic acid E, glutamic acid E and lysine K, respectively; Preferably, the general formula I selects a polypeptide having the following structure: ACV01: DISGI NASVV NIQKE IDRLN EVAKN LNESL IDLQE L, as shown in SEQ ID NO. 1; ACV02: DISGI NASVV NIQEE IDRLE EVAKN LNESL IDLQE L, as shown in SEQ ID NO. 2; ACV03: DISGI NASVV NIQEE IKRLE EVAKE LNKSL IDLQE L, as shown in SEQ ID NO. 3; More preferably, the polypeptide drug is a polypeptide with the following structure: ACV03: DISGI NASVV NIQEE IKRLE EVAKE LNKSL IDLQE L, as shown in SEQ ID NO.3, the N-terminus of the polypeptide molecule is free or connected to an amino protecting group, and the C-terminus of the polypeptide molecule is free or connected to a carboxyl protecting group.

9. The method according to claim 1, characterized in that: The reverse phase HPLC separation conditions are as follows: mobile phase A is an aqueous solution containing 0.3% formic acid, mobile phase B is acetonitrile containing 0.3% formic acid, and elution is performed according to the following procedure: 。 10. The method according to claim 1, characterized in that Tandem mass spectrometry analysis conditions are: ionization mode is ESI + , the spray voltage IS was 5500 KV, the ion source temperature TEM was 550 °C, the nebulizer gas GAS1 was 60 psi, the auxiliary nebulizer gas GAS2 was 60 psi, the curtain gas CUR was 20 psi, the collision gas CAD was 10 psi, and the scanning mode was multiple reaction monitoring MRM.

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