A method for processing and detecting a sample for detecting glucagon-like peptide in human blood

Abstract

The application discloses a processing method of a sample for detecting glucagon-like peptide in human blood, and the sample to be detected glucagon-like peptide (GLP-1) in the sample includes GLP-1 (7-36) in an active state and GLP-1 (9-36) in an inactive state. The processing of the sample includes the following steps: S1, measuring the blood sample to be detected in a clean sample tube a, adding cold acetonitrile of-20 DEG C with the same volume as the blood sample to be detected, fully mixing, standing at room temperature for precipitation, and centrifuging at 4 DEG C; S2, taking the supernatant in a clean sample tube b, adding 1% FA solution with the same volume as the supernatant, fully mixing, standing at room temperature, and centrifuging at 4 DEG C. The content of GLP-1 in the sample to be detected is obtained by drawing a standard working curve and using the detection result and the standard working curve. In the method, the signal is stronger after the sample is processed, and the GLP-1 (7-36) in the active state and the GLP-1 (9-36) in the inactive state can be simultaneously qualitatively and quantitatively analyzed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a processing and detection method of a detection sample of glucagon-like peptide in human blood. BACKGROUND

[0002] Glucagon-like peptide (GLP-1) is a polypeptide hormone released by intestinal cells after a meal, which is involved in food digestion, glucose homeostasis and body fat regulation. It plays a synergistic role in the process of blood glucose regulation, including promoting beta-cell growth, muscle sensitivity and regulating liver insulin secretion.

[0003] When GLP-1 is released into the blood circulation, it will be cleaved by dipeptidyl peptidase (DP IV) to remove the N-terminal two amino acids, and converted from GLP-1 (7-36) to GLP-1 (9-36), thereby losing its hormone regulation effect. Therefore, when GLP-1 is quantitatively detected, it is difficult to distinguish GLP-1 in active and inactive states. When collecting blood samples, attention should be paid to the inhibition of DP IV enzyme to prevent premature inhibition of GLP-1 activity.

[0004] Traditional detection methods mainly include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). Comprehensive analysis of the existing GLP-1 detection methods has the following defects and deficiencies:

[0005] HPLC method for determining GLP-1 only has quantitative results, cannot be qualitative, is easily disturbed by other substances in complex plasma samples, and thus the quantitative results are not accurate. In addition, ELISA method is currently mainly used for scientific research, is not suitable for clinical diagnosis, and is expensive. Furthermore, ELISA method has low specificity for determining different truncated GLP-1 polypeptides. For example, there is obvious cross reaction between complete GLP-1 (1-36) and GLP-1 (9-36) or GLP-1 (7-36) polypeptides.

[0006] Radioimmunoassay (RIA) for determining GLP-1 is simple and fast, but has radioactivity, which is harmful to the body.

[0007] It has been reported in the literature that mass spectrometry is used to determine GLP-1 in plasma, and high-concentration inorganic acid phosphoric acid is used in the pretreatment process, which has a great damage to the mass spectrometer. SUMMARY

[0008] In view of the existing technical problems, the present application aims to provide an improved sample processing and detection method for detecting glucagon-like peptide in human blood, which simultaneously determines GLP-1(7-36) and GLP-1(9-36) in blood plasma by using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0009] In order to achieve the above-mentioned purpose, the present application adopts the following technical means:

[0010] The first aspect of the present application provides a sample processing method for detecting glucagon-like peptide in human blood, wherein the glucagon-like peptide (GLP-1) to be detected in the detection sample includes GLP-1(7-36) in active state and GLP-1(9-36) in inactive state, and the processing of the detection sample includes the following steps:

[0011] S1, measuring the blood sample to be detected in a clean sample tube a, adding an equal volume of -20℃ cold acetonitrile to the blood sample to be detected, thoroughly mixing, standing at room temperature for precipitation, and centrifuging at 4℃.

[0012] S2, taking the supernatant in a clean sample tube b, adding an equal volume of 1% FA solution to the supernatant, thoroughly mixing, standing at room temperature, and centrifuging at 4℃.

[0013] Further, the sample purification step is also included, and the sample purification adopts an SPE column, and the purification process is carried out under the condition of 0.04mPa nitrogen gas pressure filtration: the SPE column is activated by repeatedly passing acetonitrile solution twice; the SPE column is balanced by repeatedly passing ultrapure water twice; the sample is passed through the SPE column; the SPE column is washed by repeatedly passing ultrapure water twice; and the SPE column is passed through 70% acetonitrile water solution and the filtrate is recovered, and the filtrate is dried under high flow rate nitrogen gas at 45℃ to obtain the purified detection sample.

[0014] Preferably, the GLP-1(9-36) internal standard working solution is added to the detection sample.

[0015] Further, the concentration of the GLP-1(9-36) internal standard working solution is 5000ng / mL, which is prepared from GLP-1(9-36) internal standard and blank matrix.

[0016] The second aspect of the present application provides a detection method for glucagon-like peptide in human blood, which detects the detection sample described above by using HPLC-MS / MS, and includes the following steps:

[0017] (1) GLP-1 (7-36) standard solution was prepared by using GLP-1 (7-36) standard and blank matrix, and GLP-1 (9-36) standard solution was prepared by using GLP-1 (9-36) standard and blank matrix, and the concentrations of GLP-1 (7-36) standard solution and GLP-1 (9-36) standard solution were the same;

[0018] (2) GLP-1 standard curve working solution with designed concentration gradient was prepared by mixing equal amounts of GLP-1 (7-36) standard solution and GLP-1 (9-36) standard solution, and then diluting with blank matrix;

[0019] (3) Equal amounts of GLP-1 (9-36) internal standard working solution were added to each concentration of GLP-1 standard curve working solution, and after HPLC-MS / MS detection, the corresponding standard working curve was drawn with the concentration of standard curve working solution as the horizontal coordinate and the ratio of GLP-1 (7-36) and GLP-1 (9-36) quantitative ion mass spectrum peak area to GLP-1 (9-36) internal standard quantitative ion mass spectrum peak area as the vertical coordinate;

[0020] (4) The treated detection samples were detected by HPLC-MS / MS, and the concentrations of GLP-1 (7-36) and GLP-1 (9-36) in the detection samples were calculated by using the detection results and the corresponding standard working curve.

[0021] Further, the blank matrix is 2% BSA.

[0022] Further, the chromatographic conditions of HPLC-MS / MS detection are as follows:

[0023] Chromatographic column: Phenomenex Kinetex 2.6 μm C8 LC Column (50x2.1mm);

[0024] Mobile phase: mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile solution;

[0025] Flow rate: 0.3 mL / min;

[0026] Column temperature: 45℃;

[0027] Injection volume: 15 μL;

[0028] Gradient elution mode was used, and the gradient elution mode was as follows: volume fraction of mobile phase A + volume fraction of mobile phase B = 100%; gradient elution time 8 min stop, wherein:

[0029] 0-0.4 min, volume fraction of mobile phase A was 95%;

[0030] 0.4-0.5 min the volume fraction of mobile phase A is decreased from 95% to 72%;

[0031] 0.5-4 min the volume fraction of mobile phase A is decreased from 72% to 62%;

[0032] 4-4.1 min the volume fraction of mobile phase A is decreased from 62% to 2%;

[0033] 4.1-6 min the volume fraction of mobile phase A is kept at 2%;

[0034] 6-6.1 min the volume fraction of mobile phase A is increased from 2% to 95%;

[0035] 6.1-7.9 min the volume fraction of mobile phase A is kept at 95%.

[0036] Further, the mass spectrometry conditions for HPLC-MS / MS detection are as follows: ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature: 500 °C; ion source nebulizer gas: 55 psi; ion source heated auxiliary gas: 55 psi; gas curtain gas: 40 psi; collision gas: Medium; scan mode: Scheduled MRM,

[0037] The mass spectrometry parameters for GLP-1 (7-36) are as follows: quantitation ion, 7-36_3+_y20; Q1, 660.300 (m / z); Q3, 752.300 (m / z); RT, 2.4 (min); DP, 80 (V); EP, 10 (V); CE, 25 (V); CXP, 25 (V);

[0038] The mass spectrometry parameters for GLP-1 (9-36) are as follows: quantitation ion, 9-36_3+_y23; Q1, 773.100 (m / z); Q3, 852.500 (m / z); RT, 2.8 (min); DP, 80 (V); EP, 10 (V); CE, 25 (V); CXP, 26 (V);

[0039] The mass spectrometry parameters for GLP-1 (9-36) internal standard are as follows: quantitation ion, 9-36_H_3+_y23; Q1, 775.800 (m / z); Q3, 855.777 (m / z); RT, 2.8 (min); DP, 80 (V); EP, 10 (V); CE, 25 (V); CXP, 26 (V).

[0040] Advantages of the present application

[0041] Compared with the prior art, the present application has the following advantages:

[0042] 1. The method for detecting GLP-1 in blood plasma by HPLC-MS / MS is optimized and improved, the mass-to-charge ratio (m / z) of a parent ion and a secondary fragment ion is monitored by a multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, high sensitivity and specific quantitative determination of GLP-1 is realized; qualitative and quantitative analysis of GLP-1 (7-36) in an active state and GLP-1 (9-36) in an inactive state can be simultaneously performed, and the level of GLP-1 in a blood plasma sample is comprehensively evaluated.

[0043] 2. In the sample pretreatment process, the method of adopting phosphoric acid treatment in the prior art is changed to formic acid treatment, on the one hand, the mass spectrometer is not damaged, on the other hand, the response signal of GLP-1 is significantly enhanced.

[0044] 3. The ratio of the precipitant to the sample makes the effect of precipitating protein better, meanwhile, GLP-1 in blood plasma can not be precipitated, a good recovery rate can be achieved, and the pollution of macromolecular protein to the instrument is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 A spectrum of a detection sample in condition 1 in Example 1 is shown;

[0046] Figure 2 A spectrum of a detection sample in condition 2 in Example 2 is shown;

[0047] Figure 3 A detection result of a standard curve working solution in condition 1 in Example 3 is shown;

[0048] Figure 4 A detection result of a standard curve working solution in condition 2 in Example 4 is shown;

[0049] Figure 5 An ion flow chart of GLP-1 (7-36) in Example 5 is shown;

[0050] Figure 6 An ion flow chart of GLP-1 (9-36) in Example 5 is shown;

[0051] Figure 7 An ion flow chart of GLP-1 (9-36) in Example 5 is shown.

[0052] Figure 8 A standard working curve chart of GLP-1 (7-36) in Example 5 is shown;

[0053] Figure 9 A standard working curve chart of GLP-1 (9-36) in Example 5 is shown;

[0054] Figure 10The spectrum of GLP-1(7-36) sample detection in Example 5 is shown;

[0055] Figure 11 The spectrum of GLP-1(9-36) sample detection in Example 5 is shown; Detailed Implementation

[0056] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions disclosed in these documents concerning synthetic techniques, product and processing design, polymers, comonomers, initiators, or catalysts in the art. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0057] The numerical ranges in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. A numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For example, if the stated composition, physical, or other property (such as molecular weight, melt index, etc.) is 100 to 1000, it means that all individual values, such as 100, 101, 102, etc., are explicitly listed, as well as all subranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly stated in this application.

[0058] When referring to chemical compounds, unless explicitly stated otherwise, the singular includes all isomers and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). Additionally, unless explicitly stated otherwise, nouns described with "an," "a," or "the" also include their plural forms.

[0059] The terms "comprising," "including," "containing," and "having," and their derivatives, are not intended to exclude any component, step or procedure not specified, and are used synonymously with "including" or "having" unless otherwise indicated. For the avoidance of doubt, unless expressly stated otherwise, all compositions comprising a term "comprising," "including," or "having" as used herein, can include any additional additive, adjuvant, or compound. In contrast, the term "consisting essentially of limits any other components, steps or procedures to those that do not materially affect the basic and novel characteristic(s) of the compositions or methods described in the present disclosure. The term "consisting of excludes any component, step or procedure not specifically recited. The term "or" as used herein is to be interpreted as inclusive or meaning any one or any combination. Therefore, "A, B or C" means "any of the following: A; B; or C; A and B; A and C; B and C; or A, B and C."

[0060] In order to make the technical problems solved by the present application, technical solutions and beneficial effects clearer, the present application will be further described in detail below in combination with embodiments.

[0061] I. Instruments and equipment

[0062] Sciex 5500 QT triple quadrupole mass spectrometer (USA, Sciex Corporation), including Shimadzu high-performance liquid chromatography system (Japan, Shimadzu Scientific Company);

[0063] KQ-500E ultrasonic cleaner (China, Kunshan Ultrasonic Instrument Co., Ltd.);

[0064] 1-14KS / 3-18KS benchtop high-speed refrigerated centrifuge (Germany, Sigma);

[0065] EVortex-Genie2 vortex mixer (USA, Scientific Industries Company);

[0066] Cascada I ultrapure water preparation system (Canada, Pall Forti Biological Analysis Instrument (Shanghai) Co., Ltd.);

[0067] QUINTIX 125D-1CN electronic balance (Germany, Sartorius Scientific Instruments (Beijing) Co., Ltd.);

[0068] MD200-2 centrifuge tube nitrogen blow concentration device (China, Hangzhou Aosheng Instrument Co., Ltd.).

[0069] II. Reagents and consumables

[0070] Standard: Glucagon-like peptide GLP-1 (7-36), 5 mg, divided into 5 tubes.

[0071] Sequence: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR

[0072] Glucagon-like peptide GLP-1(9-36), 5 mg, divided into 5 tubes.

[0073] Sequence: EGTFTSDVSSYLEGQAAKEFIAWLVKGR

[0074] Chromatographic acetonitrile purchased from Merck (Germany)

[0075] Chromatographic grade formic acid purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (China);

[0076] Bovine serum albumin purchased from Biyun Tian Company (China);

[0077] Plasma samples were from plasma samples sent by Hangzhou Du'an Medical Laboratory Co., Ltd;

[0078] Centrifuge tube 1.5 mL (Axygen, Germany);

[0079] 300 μL 96-well plate and sealing gasket purchased from Biosepur Company;

[0080] Biocomma HLB SPE 96-well plate, Comma Company.

[0081] III. Preparation of reagents

[0082] Preparation of blank matrix (2% bovine serum albumin): 2 g of BSA was prepared in 100 mL of ultrapure water (the volume of BSA was configured according to the sample amount, and the remaining volume was stored in a 4°C refrigerator)

[0083] Cold acetonitrile: 100% acetonitrile was placed in a -20°C refrigerator for standby.

[0084] 1% FA solution preparation: 200 μL of 100% FA solution was taken by pipette, added to 19.800 mL of ultrapure water, and mixed well. Standby at room temperature.

[0085] 4% phosphoric acid solution preparation: 940 μL of 85% phosphoric acid solution was taken by pipette, added to 19.06 mL of ultrapure water, and mixed well. Standby at room temperature.

[0086] IV. HPLC-MS / MS detection conditions

[0087] (1) Chromatographic conditions

[0088] Chromatographic column: Phenomenex Kinetex 2.6 μm C8 LC Column (50 x 2.1 mm);

[0089] Mobile phase: mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile;

[0090] Flow rate: 0.3 mL / min;

[0091] Column temperature: 45°C;

[0092] Injection volume: 15 μL; gradient elution was used, and the gradient elution was as shown in Table 1:

[0093] Table 1 Elution gradient

[0094]

[0095] (2) Mass spectrometry conditions

[0096] Ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature (TEM): 500°C; ion source atomization gas (GS1): 55 psi; ion source heating auxiliary gas (GS2): 55 psi; gas curtain gas (CUR): 40 psi; collision gas (CAD): Medium; scan mode: Scheduled MRM (Enabled), and the mass spectrometry parameters are shown in Table 2.

[0097] Table 2 MRM quantitative detection ion pair

[0098] Quantitation ion Q1 (m / z) Q3 (m / z) RT (min) DP (V) EP (V) CE (V) CXP (V) 7-36_3+_y20 660.300 752.300 2.4 80 10 25 25 9-36_3+_y23 773.100 852.500 2.8 80 10 25 26 9-36_H_3+_y23 775.800 855.777 2.8 80 10 25 26

[0099] In the above table, 7-36_3+_y20 is the GLP-1 (7-36) quantitative ion; 9-36_3+_y23 is the GLP-1 (9-36) quantitative ion; 9-36_H_3+_y23 is the GLP-1 (9-36) internal standard quantitative ion; Q1: parent ion; Q3: daughter ion; RT: retention time; DP: declustering voltage; EP: entrance voltage; CE: collision energy; CXP: collision cell exit voltage.

[0100] Examples 1-2

[0101] 1. Sample pretreatment

[0102] S1, 1.5 mL centrifuge tubes were used to prepare the samples; 300 μL of human plasma was accurately measured into two tubes, which were placed in clean centrifuge tubes; 3 μL of GLP-1 (9-36) internal standard working solution was added, and mixed;

[0103] S2, Example 1: 161 μL of cold acetonitrile was added to the tube; Example 2: 300 μL of cold acetonitrile was added to the tube, and mixed thoroughly, and then left to stand at room temperature for 3 minutes; centrifugation was performed at 13000 g at 4°C for 10 min (the centrifuge was pre-cooled);

[0104] S3, 400 μL supernatant was taken into a new 1.5 mL centrifuge tube, 400 μL 1% FA solution was added, mixed well, and placed at room temperature for 3 minutes; centrifuged at 13000 g at 4°C for 10 minutes (the centrifuge was pre-cooled);

[0105] 2, SPE purification of sample

[0106] S1, 500 μL acetonitrile solution was added to the SPE column, and the column was activated by filtering under 0.04 mPa nitrogen pressure, and the operation was repeated twice;

[0107] S2, 500 μL ultrapure water was added, and the column was equilibrated by filtering under 0.04 mPa nitrogen pressure, and the operation was repeated twice;

[0108] S3, 700 μL sample was added, and the column was filtered under 0.04 mPa nitrogen pressure;

[0109] S4, 500 μL ultrapure water was added, and the column was washed by filtering under 0.04 mPa nitrogen pressure, and the operation was repeated twice;

[0110] S5, 200 μL 70% acetonitrile aqueous solution was added, and the filtrate was recovered by filtering under 0.04 mPa nitrogen pressure;

[0111] S6, the filtrate was transferred to a 1.5 mL centrifuge tube, and the solvent was evaporated by high-flow nitrogen gas at 45°C for about 2 hours, and the sample was stored in a -80°C refrigerator for standby.

[0112] 3, HPLC-MS / MS detection

[0113] The treated sample was detected on the HPLC-MS / MS, the sample amount was 15 μL, and the chromatographic and mass spectrometric parameters were set as described above. The detection results of Example 1 and Example 2 are shown in Figure 1 and Figure 2 .

[0114] The results show that the peak type and response of GLP-1(9-36) are very different when the ratio of the precipitant in S2 is different. As can be seen from the results, when the ratio of the sample to the precipitant is 1.86:1, there is basically no peak; when the ratio of the sample to the precipitant is adjusted to 1:1, the peak area response of GLP-1(9-36) is obviously increased, indicating that the extraction effect is better when the ratio of the sample to the precipitant is 1:1.

[0115] Examples 3-4

[0116] 1, sample pretreatment

[0117] S1, 1.5 mL centrifuge tube was used for sample preparation; 300 μL human plasma was accurately taken into two clean centrifuge tubes; 3 μL GLP-1(9-36) internal standard working solution was added, and mixed well;

[0118] S2, add 300 μL of cold acetonitrile respectively, mix well, room temperature for 3 minutes; 4 °C, 13000g centrifugation for 10 min (centrifuge in advance pre-cooling);

[0119] S3, respectively, take 400 μL supernatant in a new 1.5 mL centrifuge tube. Example 3: the tube is added with 400 μL 4% phosphoric acid solution; Example 4: the tube is added with 400 μL 1% formic acid solution, mix well, room temperature for 3 minutes; 4 °C, 13000g centrifugation for 10 min (centrifuge in advance pre-cooling);

[0120] 2, SPE purification sample

[0121] S1, add 500 μL acetonitrile solution to the SPE column, filter with 0.04 mPa nitrogen pressure, repeat twice to activate the column;

[0122] S2, add 500 μL ultrapure water, filter with 0.04 mPa nitrogen pressure, repeat twice to balance the column;

[0123] S3, add 700 μL sample, filter with 0.04 mPa nitrogen pressure;

[0124] S4, add 500 μL ultrapure water, filter with 0.04 mPa nitrogen pressure, repeat twice to rinse the column;

[0125] S5, add 200 μL 70% acetonitrile aqueous solution, filter with 0.04 mPa nitrogen pressure, and recover the filtrate;

[0126] S6, transfer all the filtrate to a 1.5 mL centrifuge tube, dry with high flow rate nitrogen at 45 °C, about 2 hours, store in a-80 °C refrigerator for standby.

[0127] 3, HPLC-MS / MS quantitative detection

[0128] The treated sample is detected on HPLC-MS / MS, the sample amount is 15 μL, the chromatographic and mass spectrometric parameters are set as described above, and the detection results of example 3 and example 4 are shown in the following Figure 3 and Figure 4 .

[0129] Comparison Figure 3 and Figure 4 It can be seen that after replacing 4% phosphoric acid solution with 1% formic acid solution, the peak area response is enhanced by 10 times, and the linearity is better Figure 3 and Figure 4 The red in the table shows that it does not meet the standard, Figure 4 the linearity is better), and the minimum quantitative point concentration of 1% formic acid solution is lower.

[0130] Example 5

[0131] The final experimental conditions were determined by integrating Examples 1 to 4, i.e. the sample to precipitant ratio was set to 1:1; the 4% phosphoric acid solution was replaced with a 1% formic acid solution, and the specific implementation is as follows.

[0132] 1. Sample pretreatment

[0133] S1, a 1.5 mL centrifuge tube was used to prepare the sample; 300 μL of human blood plasma was accurately measured and placed in a clean centrifuge tube; 3 μL of GLP-1 (9-36) internal standard working solution was added and mixed well;

[0134] S2, 300 μL of cold acetonitrile was added and mixed well, and it was left to stand at room temperature for 3 minutes; 4°C, 13000g centrifugation for 10 min (the centrifuge was pre-cooled in advance);

[0135] S3, 400 μL of supernatant was taken in a new 1.5 mL centrifuge tube, 400 μL of 1% FA solution was added, mixed well, and left to stand at room temperature for 3 minutes; 4°C, 13000g centrifugation for 10 min (the centrifuge was pre-cooled in advance);

[0136] 2. SPE purified sample

[0137] S1, 500 μL of acetonitrile solution was added to the SPE column, and 0.04 mPa nitrogen gas was used to pressurize and filter, and the column was activated twice;

[0138] S2, 500 μL of ultrapure water was added, and 0.04 mPa nitrogen gas was used to pressurize and filter, and the column was equilibrated twice;

[0139] S3, 700 μL of sample was added, and 0.04 mPa nitrogen gas was used to pressurize and filter;

[0140] S4, 500 μL of ultrapure water was added, and 0.04 mPa nitrogen gas was used to pressurize and filter, and the column was washed twice;

[0141] S5, 200 μL of 70% acetonitrile aqueous solution was added, and 0.04 mPa nitrogen gas was used to pressurize and filter, and the filtrate was recovered;

[0142] S6, the filtrate was transferred to a 1.5 mL centrifuge tube, and the nitrogen gas was blown dry at 45°C at a high flow rate for about 2 hours, and it was stored in a -80°C refrigerator for standby.

[0143] 4. Preparation of GLP-1 standard curve

[0144] (1) Preparation of standard mother liquor

[0145] Blank matrix: bovine serum albumin was dissolved and mixed with ultrapure water to prepare a 2% BSA solution.

[0146] Take GLP-1 (7-36) standard 1 mg, add 1 mL of blank matrix for dissolution, get GLP-1 (7-36) stock solution 1 μg / μL, stored in-80℃ refrigerator for standby;

[0147] Take GLP-1 (9-36) standard 1 mg, add 1 mL of blank matrix for dissolution, get GLP-1 (7-36) stock solution 1 μg / μL, stored in-80℃ refrigerator for standby;

[0148] (2) GLP-1 standard working solution preparation

[0149] Take GLP-1 (7-36) stock solution and GLP-1 (9-36) stock solution 1 μL each, mix the stock solution dilution 1000 times, get mixed standard R0 solution, concentration is 1000 ng / mL;

[0150] Take 200 μL R0 solution and add 1800 μL of blank matrix solution, get W0 solution, concentration is 100 ng / mL;

[0151] GLP-1 (7-36) standard curve points W1-W8 are diluted by W0 solution with blank matrix, the concentration and preparation of standard curve working solution W1-W9 are shown in table 3;

[0152] GLP-1 (9-36) standard curve points W1-W8 are diluted by W0 solution with blank matrix, the concentration and preparation of standard curve working solution W1-W8 are shown in table 4.

[0153] Table 3 GLP-1 (7-36) standard curve working solution concentration

[0154] Standard curve working solution name Concentration (ng / mL) Volume (μL) pipetted Blank matrix (μL) W1 10 200 1800 W2 7.5 150 1850 W3 5 100 1900 W4 2 40 1960 W5 1 20 1980 Standard curve working solution name Concentration (ng / mL) Volume (μL) pipetted Blank matrix (μL) W6 0.5 500 1500 W7 0.1 100 1900 W8 0.05 50 1950

[0155] Table 4 GLP-1 (9-36) standard curve working solution concentration

[0156] Standard curve working solution name Concentration (ng / mL) Volume (μL) pipetted Blank matrix (μL) W1 75 1500 500 W2 50 1000 1000 W3 25 500 1500 W4 10 200 1800 W5 7.5 150 1850 W6 5 100 1900 W7 2 40 1960 W8 1 20 1980

[0157] 5、HPLC-MS / MS quantitative detection

[0158] GLP-1 (9-36) internal standard stock solution: take GLP-1 (9-36) internal standard stock solution 1000 μg, add 1000 μL of blank matrix for dissolution, mix well to get GLP-1 (9-36) internal standard stock solution 1 μg / μL, aliquot and store in-80℃ refrigerator.

[0159] GLP-1(9-36) internal standard working solution: 1 μL of internal standard stock solution was diluted with 199 μL of blank matrix, and mixed to obtain GLP-1(9-36) internal standard working solution 5000 ng / mL.

[0160] The chromatographic and mass spectrometric parameters are shown in Table 1 and Table 2. The ion chromatogram of GLP-1(7-36) obtained by mass spectrometric detection is shown in Figure 5 ; the ion chromatogram of GLP-1(9-36) is shown in Figure 6 ; and the ion chromatogram of GLP-1(9-36) internal standard is shown in Figure 7 .

[0161] The standard curve working solution of 8 known GLP-1 concentrations of W1-W8 was prepared, 3 μL of GLP-1(9-36) internal standard working solution was added, and mass spectrometric detection was performed. The concentration of the standard curve working solution was taken as the abscissa, and the ratio of the mass spectrometric peak area of the GLP-1(7-36) and GLP-1(9-36) quantification ion to the mass spectrometric peak area of the GLP-1(9-36) internal standard quantification ion was taken as the ordinate, and the corresponding standard working curve was drawn, and the corresponding linear equation was obtained as follows:

[0162] The linear equation of GLP-1(7-36) is as follows: y=0.00769x+2.30556e -4 (r=0.99082); and the standard working curve is shown in Figure 8 .

[0163] The linear equation of GLP-1(9-36) is as follows: y=0.00871x+0.00928 (r=0.99759); and the standard working curve is shown in Figure 9 .

[0164] The treated sample was detected on the HPLC-MS / MS, and the injection amount was 15 μL. The sample chromatograms of GLP-1(7-36) and GLP-1(9-36) are shown in Figure 10 and Figure 11 , and the sample detection results are shown in Table 5.

[0165] Table 5 Sample detection results

[0166]

[0167] All the documents mentioned in the present application are incorporated by reference. In addition, it should be understood that various modifications and changes can be made to the present application by those skilled in the art upon reading the above description of the present application, and these equivalent forms also fall within the scope of the appended claims of the present application.

Claims

1. A method for processing a sample for detecting a glucagon-like peptide in human blood, characterized by: The detection sample includes active GLP-1 (7-36) and inactive GLP-1 (9-36), and the processing of the detection sample includes the following steps: S1, measuring the blood sample in a clean sample tube a, adding an equal volume of -20℃ cold acetonitrile, mixing thoroughly, standing at room temperature, precipitating, and centrifuging at 4℃; S2, taking the supernatant in a clean sample tube b, adding an equal volume of 1% FA solution, mixing thoroughly, standing at room temperature, and centrifuging at 4℃; The sample purification process is performed under the condition of 0.04 mPa nitrogen pressure filtration: the SPE column is activated by repeatedly passing acetonitrile solution twice; the SPE column is balanced by repeatedly passing ultrapure water twice; the sample is passed through the SPE column; the SPE column is washed by repeatedly passing ultrapure water twice; and the filtrate is recovered after passing the SPE column with 70% acetonitrile water solution, and the filtrate is dried under high flow rate nitrogen at 45℃ to obtain the purified detection sample. The sample purification is performed by using Biocomma HLB SPE 96-well plate.

2. The method for processing a sample for detecting glucagon-like peptide in human blood according to claim 1, characterized in that: The GLP-1 (9-36) internal standard working solution is added to the detection sample.

3. The method for processing a sample for detecting glucagon-like peptide in human blood according to claim 2, characterized in that: The GLP-1 (9-36) internal standard working solution has a concentration of 5000 ng / mL and is prepared by using GLP-1 (9-36) internal standard and blank matrix.

4. A method for detecting glucagon-like peptide in human blood, characterized by: The detection method is to detect the detection sample of claim 3 by using HPLC-MS / MS, including the following steps: (1) preparing GLP-1 (7-36) standard mother liquor by using GLP-1 (7-36) standard and blank matrix, and preparing GLP-1 (9-36) standard mother liquor by using GLP-1 (9-36) standard and blank matrix, the concentrations of GLP-1 (7-36) standard mother liquor and GLP-1 (9-36) standard mother liquor are the same; (2) mixing equal amounts of GLP-1 (7-36) and GLP-1 (9-36) standard mother liquor, and then diluting with blank matrix to prepare GLP-1 (7-36) and GLP-1 (9-36) standard curve working solution with designed concentration gradient; (3) adding equal amounts of GLP-1 (9-36) internal standard working solution to each GLP-1 standard curve working solution, detecting by HPLC-MS / MS, taking the concentration of standard curve working solution as the horizontal coordinate, and taking the ratio of GLP-1 (7-36) and GLP-1 (9-36) quantitative ion mass spectrum peak area to GLP-1 (9-36) internal standard quantitative ion mass spectrum peak area as the vertical coordinate, to draw the corresponding standard working curve; (4) detecting the processed detection sample by using HPLC-MS / MS, and calculating the concentrations of GLP-1 (7-36) and GLP-1 (9-36) in the detection sample by using the detection result and the corresponding standard working curve.

5. The method for detecting glucagon-like peptide in human blood according to claim 4, characterized by: The blank matrix is 2% BSA.

6. The method for detecting glucagon-like peptide in human blood according to claim 4, characterized by: The chromatographic conditions of HPLC-MS / MS detection are as follows: Column: Phenomenex Kinetex 2.6 μm C8 LC Column, 50 × 2.1 mm; Mobile phase: mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile; Flow rate: 0.3 mL / min; Column temperature: 45℃; Injection volume: 15 μL; The gradient elution method was used, and the gradient elution method was as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; stop at 8 min of gradient elution time, wherein: 0-0.4 min, the volume fraction of mobile phase A was 95%; 0.4-0.5 min, the volume fraction of mobile phase A decreased from 95% to 72%; 0.5-4 min, the volume fraction of mobile phase A decreased from 72% to 62%; 4-4.1 min, the volume fraction of mobile phase A decreased from 62% to 2%; 4.1-6 min, the volume fraction of mobile phase A remained at 2%; 6-6.1 min, the volume fraction of mobile phase A increased from 2% to 95%; 6.1-7.9 min, the volume fraction of mobile phase A remained at 95%.

7. The method for detecting glucagon-like peptide in human blood according to claim 4, characterized by: The mass spectrometry conditions for HPLC-MS / MS detection were as follows: Ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature: 500℃; ion source atomization gas: 55 psi; ion source heating auxiliary gas: 55 psi; Gas curtain: 40 psi; Collision gas: Medium; scan mode: Scheduled MRM; The mass spectrometry parameters for GLP-1 (7-36) were as follows: quantitative ion, 7-36_3+_y20; Q1, 660.300 m / z; Q3, 752.300 m / z; RT, 2.4 min; DP, 80 V; EP, 10 V; CE, 25 V; CXP, 25 V; The mass spectrometry parameters for GLP-1 (9-36) were as follows: quantitative ion, 9-36_3+_y23; Q1, 773.100 m / z; Q3, 852.500 m / z; RT, 2.7 min; DP, 80 V; EP, 10 V; CE, 25 V; CXP, 26 V; The mass spectrometry parameters for GLP-1 (9-36) internal standard were as follows: quantitative ion, 9-36_H_3+_y23; Q1, 775.800 m / z; Q3, 855.777 m / z; RT, 2.7 min; DP, 80 V; EP, 10 V; CE, 25 V; CXP, 26 V.

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