A method for processing and detecting samples of insulin-like growth factor in blood

Abstract

This invention discloses a method for processing and detecting insulin-like growth factor (IGF) in blood samples. The sample is treated with a 0.6% SDS dissociator and precipitant, and the precipitate is further washed with glacial acetonitrile to remove SDS and reduce damage to the instrument. Detection is performed using established chromatographic and mass spectrometry conditions, and a standard curve is used to determine the IGF content in the blood sample. This method does not require SPE purification, and mass spectrometry detection takes only 4.5 minutes, significantly reducing costs and detection time for large sample volume applications.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a method for processing and detecting a sample of insulin-like growth factor in blood. Background Art

[0002] Insulin-like growth factor (IGF-1) is a polypeptide hormone composed of 70 amino acids with a molecular weight of approximately 7.6kDa. It is secreted from the liver in response to growth hormone (GH). Therefore, IGF-1 is often used as a supplement or alternative to GH. In the blood, IGF-1 typically exists as a complex with IGF-binding proteins (IGFBPs).

[0003] Traditional detection methods, primarily enzyme-linked immunosorbent assays (ELISAs), have low sensitivity and are susceptible to interference from other analogs such as IGF-2. With the rapid development of clinical mass spectrometry in recent years, mass spectrometry has also rapidly gained acceptance for IGF-1 detection. However, IGF-1's large molecular weight and presence in the form of a protein complex in the blood make accurate quantification of IGF-1 challenging.

[0004] Comprehensive analysis shows that the current IGF-1 detection methods have the following defects and shortcomings:

[0005] 1. Enzyme-linked immunosorbent assay: This detection method has low sensitivity and cannot accurately quantify the clinical diagnosis of dwarfism in children.

[0006] 2. Trypsin digestion: Due to the large molecular weight of IGF-1, it requires enzymatic digestion to detect the peptide fragments. This mass spectrometry method is time-consuming and generally requires overnight processing. Furthermore, the need for trypsin treatment increases testing costs.

[0007] 3. Intact protein detection using a dissociation agent: Because IGF-1 exists as a protein complex, a dissociation agent is required. Current dissociation agents primarily include sodium dodecyl sulfate (SDS) and trifluoroethanol (TFE). These agents significantly interfere with mass spectrometry and easily contaminate the mass spectrometer. Pre-treated samples must undergo SPE purification to remove SDS, significantly increasing the cost of the assay.

[0008] 4. Adding IGF-2 to serum to compete for dissociation and obtain complete IGF-1: This method requires the additional addition of IGF-2, which increases the detection cost, and the dissociation effect of IGF-1 is not good. Summary of the Invention

[0009] In view of the technical problems existing in the background technology, the purpose of the present invention is to provide a method for detecting insulin-like growth factor in blood.

[0010] In order to achieve the above object, the present invention adopts the following technical means:

[0011] A first aspect of the present invention is to provide a method for processing a sample for detecting insulin-like growth factor in blood, comprising the following steps:

[0012] S1. Measure the sample to be tested and place it in a clean centrifuge tube a. Add 0.6% SDS in an equal volume to the sample to be tested, mix thoroughly, centrifuge the solution to the bottom of the centrifuge tube, and incubate at 37°C for 20-40 minutes.

[0013] S2. Add precipitant consisting of acetic acid, acetonitrile, and acetone twice the volume of the solution in centrifuge tube a, mix thoroughly, precipitate the protein for 1-3 minutes, and centrifuge at 4°C.

[0014] S3. Pipette the supernatant into a clean centrifuge tube b, add 4.2 times the volume of cold acetonitrile, mix thoroughly, and place in a -20°C refrigerator for protein precipitation;

[0015] S4. Remove the protein precipitate sample from the -20°C freezer and centrifuge at 4°C, discard the supernatant; add an appropriate amount of cold acetonitrile to the precipitate, mix thoroughly, wash the precipitate and the wall of the centrifuge tube, centrifuge at 4°C, discard the supernatant, and air dry;

[0016] S5. Add 0.1% formic acid aqueous solution, vortex and shake to fully dissolve to obtain the test sample.

[0017] Furthermore, the precipitant is mixed in a volume ratio of acetic acid:acetonitrile:acetone=1:4:15.

[0018] The second aspect of the present invention provides a method for detecting insulin-like growth factor in blood samples based on HPLC-MS / MS, comprising the following steps: preparing a standard curve working solution with a designed concentration gradient using an IGF-1 standard and a blank matrix, detecting the standard curve working solution using HPLC-MS / MS, adopting an external standard quantification method, with the concentration of the standard curve working solution as the X-axis and the chromatographic peak area of ​​the standard curve working solution as the Y-axis, to establish a standard working curve; injecting the test sample into the HPLC-MS / MS for measurement, and substituting the detected sample chromatographic peak area into the standard working curve to obtain the IGF-1 concentration in the test sample.

[0019] Furthermore, the blank matrix is ​​a 2% BSA solution.

[0020] Preferably, the chromatographic conditions for the detection are as follows:

[0021] Chromatographic column: Phenomenex Kinetex C8 column (50x2.1 mm, 2.6 μm, 100 Å);

[0022] Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile;

[0023] Flow rate: 0.4 mL / min;

[0024] Column temperature: 55°C;

[0025] Injection volume: 15 μL

[0026] A 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%; the gradient elution time was 4.5 minutes and then stopped, wherein:

[0027] 0.01-0.5min The volume fraction of mobile phase A is 95%;

[0028] The volume fraction of mobile phase A decreased from 95% to 80% at 0.5-0.6 min;

[0029] The volume fraction of mobile phase A decreased from 80% to 50% during 0.6-2 min;

[0030] 2-2.1 min: the volume fraction of mobile phase A decreased from 50% to 0%;

[0031] 2.1-3.1 min: the volume fraction of mobile phase A was maintained at 0%;

[0032] From 3.1 to 3.2 min, the volume fraction of mobile phase A increased and decreased from 0% to 95%;

[0033] The volume fraction of mobile phase A was maintained at 95% from 3.2 to 4.5 min.

[0034] Furthermore, the mass spectrometry conditions for the detection are as follows:

[0035] 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 heating auxiliary gas: 55 psi; curtain gas: 40 psi; collision gas: Medium; scanning mode: MRM, MRM parameters are: quantitative ion: IGF-1_8_y5; Q1 (m / z): 956.950; Q3 (m / z): 473.500; RT (min): 1.65; DP (V): 100; EP (V): 10; CE (V): 45; CXP (V): 15.

[0036] Beneficial effects of the present invention

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The present invention is based on the determination of IGF-1 concentration in human serum, and uses the denaturant SDS to incubate and dissociate IGF-1 and its binding protein IGFBPs to obtain free IGF-1. The use of 0.6% SDS has a better dissociation effect and a higher mass spectrometry detection signal value.

[0039] (2) The method of the present invention utilizes SDS to dissociate IGF-1 in serum, replacing the competitive dissociation method of adding IGF-2, thereby significantly saving detection costs.

[0040] (3) The method of the present invention uses a precipitant volume 4 times the sample volume to precipitate the protein, and then uses cold acetonitrile to perform a two-step washing precipitation to remove SDS. There is no need to use SPE for purification. The operation is simple and saves detection costs. It is suitable for the detection of IGF-1 in clinical samples and also reduces contamination to the mass spectrometer.

[0041] (4) The mass spectrometry detection method is short, only taking 4.5 minutes, which greatly saves detection time for applications with large sample volumes. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The ion current diagram of the IGF-1 quality control sample of the present invention is shown.

[0043] Figure 2 A standard working curve graph of IGF-1 is shown. DETAILED DESCRIPTION

[0044] Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages in this application are based on weight, and the test and characterization methods used are current as of the filing date of this application. Where applicable, the contents of any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, and their equivalent patent families are also incorporated by reference, especially with respect to definitions of synthetic techniques, product and processing designs, polymers, comonomers, initiators, or catalysts disclosed in these documents in the art. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0045] Numerical ranges in this application are approximate values, so unless otherwise stated, they may include numerical values ​​outside the scope. Numerical ranges include all numerical values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For example, if the description component, physical or other properties (such as molecular weight, melt index, etc.) is 100 to 1000, it means that all individual numerical values ​​are clearly enumerated, such as 100, 101, 102, etc., and all subranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For the scope comprising a numerical value less than 1 or comprising a fraction greater than 1 (such as 1.1, 1.5, etc.), 1 unit is appropriately considered as 0.0001, 0.001, 0.01 or 0.1. For the scope comprising a single digit less than 10 (such as 1 to 5), 1 unit is usually considered as 0.1. These are only specific examples of what is intended, and all possible combinations of numerical values ​​between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

[0046] When used with respect to chemical compounds, unless expressly stated otherwise, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). In addition, nouns using "a," "an," or "the" also include their plural forms unless expressly stated otherwise.

[0047] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, excipients or compounds unless expressly stated otherwise. In contrast, the term "essentially consisting of" excludes any other components, steps or processes from the scope of any subsequent description of the term, except those necessary for operational performance. The term "consisting of" does not include any components, steps or processes that are not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed members alone or in any combination.

[0048] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments.

[0049] Example

[0050] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0052] The instruments, reagents, consumables, and HPLC-MS / MS test conditions used in the following examples are as follows:

[0053] Instruments and Equipment

[0054] Sciex 5500QT triple quadrupole mass spectrometer (Sciex, USA), including Shimadzu high-performance liquid chromatography system (Shimadzu Scientific, Japan);

[0055] KQ-500E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., China);

[0056] 1-14KS / 3-18KS desktop high-speed refrigerated centrifuge (Sigma, Germany);

[0057] EVortex-Genie2 vortex mixer (Scientific Industries Company, USA);

[0058] Cascada I ultrapure water preparation system (Pall Forte Bioanalytical Instruments (Shanghai) Co., Ltd., Canada);

[0059] QUINTIX125D-1CN electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd., Germany);

[0060] MD200-2 centrifuge tube nitrogen blowing concentration device (Hangzhou Aosheng Instrument Co., Ltd., China).

[0061] Reagents and consumables

[0062] Standard: Insulin-like growth factor (IGF-1) 100 μg, purity 97%, purchased from BBI LIFE SCIENCESCORPORATION;

[0063] GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA

[0064] Chromatographic acetonitrile was purchased from Merck (Germany), chromatographic grade formic acid and glacial acetic acid (Shanghai MacLean Biochemical Technology Co., Ltd.), acetone (Shanghai Lingfeng Chemical Reagent Co., Ltd.), bovine serum albumin (Sigma), and sodium dodecyl sulfate (SigmaAldrich, USA);

[0065] 1.5 mL centrifuge tubes (Axygen, Germany);

[0066] 300 μL 96-well plates and sealing mats were purchased from Biosepur;

[0067] The plasma samples used in the experiment came from the serum samples submitted by Hangzhou Duan Medical Testing Laboratory Co., Ltd.

[0068] Reagent preparation

[0069] Preparation of blank matrix (2% bovine serum albumin): 2g BSA is prepared in 100mL ultrapure water (the volume of BSA is prepared according to the sample volume, and the remaining volume can be stored in a refrigerator at 4℃ for one week)

[0070] Preparation of dissociation agent (0.6% sodium dodecyl sulfate): 0.6 g SDS is prepared in 100 mL of ultrapure water (the volume of SDS is prepared according to the sample amount, and the remaining volume can be stored in a refrigerator at 4°C);

[0071] Preparation of precipitant (5% acetic acid, 20% acetonitrile, 75% acetone, V:V:V): Use a pipette to accurately pipette glacial acetic acid, add it to the accurately measured amounts of acetonitrile and acetone, mix thoroughly, and set aside;

[0072] Cold acetonitrile: 100% acetonitrile is placed in a -20°C refrigerator for later use.

[0073] Example 1

[0074] 1. Testing conditions

[0075] (1) Chromatographic conditions

[0076] Chromatographic column: Phenomenex Kinetex C8 column (50x2.1 mm, 2.6 μm, 100A)

[0077] Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile

[0078] Flow rate: 0.4 mL / min

[0079] Column temperature: 55°C

[0080] Injection volume: 15 μL

[0081] Gradient elution was used, and the elution gradient settings are shown in Table 1;

[0082] Table 1 IGF-1 liquid phase elution gradient

[0083] Time(min) A(％) B(％) 0.01 95 5 0.5 95 5 0.6 80 20 2.0 50 50 2.1 0 100 3.1 0 100 3.2 95 5 4.5

[0084] (2) Mass spectrometry conditions

[0085] Ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature (TEM): 500°C; ion source nebulizer gas (GS1): 55 psi; ion source heating auxiliary gas (GS2): 55 psi; curtain gas (CUR): 40 psi; collision gas (CAD): Medium; scan mode: MRM; MRM parameter conditions are shown in Table 2.

[0086] Table 2 MRM quantitative detection product ion parameters

[0087] Quantitative ion Q1(m / z) Q3(m / z) RT (min) DP(V) EP(V) CE(V) CXP(V) IGF-1_8_y5 956.950 473.500 1.65 100 10 45 15

[0088] In the above table, IGF-1_8_y5 is the quantitative ion of the IGF-1 detection sample; Q1 is the parent ion; Q3 is the product ion; RT is the retention time; DP is the declustering voltage; EP is the injection voltage, CE is the collision voltage; and CXP is the collision cell ejection voltage.

[0089] The ion current diagram of IGF-1 is as follows Figure 1 shown.

[0090] 2. IGF-1 Standard Curve

[0091] (1) Preparation of blank matrix: Weigh 2 g of bovine serum albumin, add 100 mL of ultrapure water and dissolve and mix to obtain a 2% BSA solution as the blank matrix.

[0092] (2) Preparation of IGF-1 mother solution: Take 100 μg of IGF-1 standard with a purity of 97%, add 97 μL of water to dissolve it, and obtain 1 μg / μL IGF-1 mother solution. After packaging, store it in a -80℃ refrigerator.

[0093] (3) Preparation of IGF-1 standard curve working solutions: Dilute the IGF-1 stock solution 200-fold to obtain R0 solution, with a concentration of 5000 ng / mL. Continue diluting R0 5-fold to obtain W0 solution, with a concentration of 1000 ng / mL. Dilute W0 solution with blank matrix to prepare eight different concentrations of standard curve working solutions, designated W1-W8. The concentrations and preparations of the standard curve working solutions are shown in Table 3:

[0094] Table 3 IGF-1 standard curve working solution concentration

[0095] Standard curve working solution name Concentration (ng / mL) Pipette W0 volume (μL) 2% BSA (μL) W1 750 600 200 W2 500 400 400 W3 250 200 600 W4 100 80 720 W5 50 40 760 W6 25 20 780 W7 10 8 792 W8 5 4 796

[0096] 3. Sample Processing

[0097] S1. Prepare the sample using a 1.5 mL centrifuge tube. Use a pipette to precisely measure 100 μL of human serum and place it in a clean centrifuge tube. Add 100 μL of dissociation agent and mix thoroughly. Centrifuge the solution to the bottom of the centrifuge tube and incubate at 37°C for 30 min.

[0098] S2. Add 400 μL of precipitant (5% acetic acid, 20% acetonitrile, 75% acetone, V:V:V), mix thoroughly, precipitate the protein for 2 min, and centrifuge at 13,000 g for 10 min at 4°C (pre-cool the centrifuge).

[0099] S3. Pipette 350 μL of the supernatant into a new 2 mL centrifuge tube, add 1.5 mL of pre-chilled acetonitrile, mix thoroughly, and place in a -20°C refrigerator for 1 hour to allow protein precipitation.

[0100] S4. Remove the protein precipitate sample from -20°C and centrifuge at 13,000 g for 10 min at 4°C (pre-cool the centrifuge) and discard the supernatant. Add 500 μL of cold acetonitrile to the precipitate, mix thoroughly, wash the precipitate and the walls of the centrifuge tube, centrifuge at 13,000 g for 10 min at 4°C, discard the supernatant, and air dry (try to avoid aspirating the precipitate).

[0101] S5. Use 300 μL of 0.1% formic acid aqueous solution, vortex and shake to fully dissolve to obtain the test sample.

[0102] 4. HPLC-MS / MS detection

[0103] The processed test samples were tested on an HPLC-MS / MS machine with a sample volume of 15 μL each time. The chromatographic and mass spectrometric parameters were set as described above.

[0104] Examples 2 and 3

[0105] Other experimental conditions were the same as in Example 1, with the only difference being the chromatographic columns used: a Phenomenex Kinetex C18 column (100 x 2.1 mm, 2.6 μm) and a Phenomenex Kinetex C8 column (50 x 2.1 mm, 2.6 μm). Two parallel experiments were performed using the same sample a. The test results are shown in Table 4 below.

[0106] Table 4 Comparison of chromatographic column results

[0107]

[0108] The results showed that through parallel experiments, the peak area response of C8 column was significantly enhanced compared with that of C18 column.

[0109] Examples 4, 5, 6, and 7

[0110] Other experimental conditions were the same as in Example 1, except that the dissociation agents used were 150 μL 50% TFE, 150 μL 100% TFE, 100 μL 0.6% SDS, and 100 μL 0.3% SDS, respectively. The same sample b was used for the experiments, and the test results are shown in Table 5 below.

[0111] Table 5 Comparison of dissociation agent results

[0112]

[0113] The results showed that the dissociation effect of TFE was poor and it also had a certain impact on the mass spectrometer; the peak area of ​​the dissociation agent 0.6% SDS was higher than that of other dissociation agents.

[0114] Examples 8 to 13

[0115] Other experimental conditions were the same as in Example 1, except that the precipitants were: 400 μL of 5% acetic acid, 95% acetonitrile; 400 μL of 5% acetic acid, 20% acetonitrile, 75% acetone; 400 μL of 100% acetonitrile; 400 μL of 25% acetonitrile, 75% acetone; 600 μL of 5% acetic acid, 95% acetonitrile; and 600 μL of 5% acetic acid, 20% acetonitrile, 75% acetone. Experiments were conducted using the same sample c, and the test results are shown in Table 6 below.

[0116] Table 6 Comparison of precipitant results

[0117]

[0118] The results showed that the peak area of ​​the sample was the highest when a precipitant with a volume 4 times that of the sample was used for precipitation, and the precipitant system was 5% acetic acid, 20% acetonitrile, and 75% acetone.

[0119] result:

[0120] The eight standard curve working solutions of W1 to W8 with known IGF-1 concentrations prepared in Example 1 were tested. The chromatographic and mass spectrometric parameters were set as described above. The external standard quantification method was used, with the concentration of the standard curve working solution as the X-axis and the chromatographic peak area of ​​the standard curve working solution as the Y-axis to establish a standard curve. The corresponding standard curve equation was: y = 102.36796x - 40.91264 (R = 0.99948). The standard curve is shown in the figure below. Figure 2 shown.

[0121] The treated test samples in Example 1 were tested on an HPLC-MS / MS machine, with a sample volume of 15 μL each time. The final results are shown in Table 7 below:

[0122] Table 7 Sample test results

[0123] sample Sample peak area IGF-1 concentration (ng / ml) serum 1.17E+04 114.447

[0124] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for detecting insulin-like growth factor in blood based on HPLC-MS / MS, characterized in that: The method comprises the following steps: preparing a standard curve working solution with a designed concentration gradient using an IGF-1 standard substance and a blank matrix, detecting the standard curve working solution using HPLC-MS / MS, adopting an external standard quantification method, with the concentration of the standard curve working solution as the X-axis and the chromatographic peak area of ​​the standard curve working solution as the Y-axis, to establish a standard working curve; injecting a test sample into the HPLC-MS / MS for measurement, and substituting the detected sample chromatographic peak area into the standard working curve to obtain the IGF-1 concentration in the test sample; The method for processing the test sample comprises the following steps: S1. Measure the sample to be tested and place it in a clean centrifuge tube a. Add 0.6% SDS in an equal volume to the sample to be tested, mix thoroughly, centrifuge the solution to the bottom of the centrifuge tube, and incubate at 37°C for 20-40 minutes. S2. Add precipitant consisting of acetic acid, acetonitrile, and acetone twice the volume of the solution in centrifuge tube a, mix thoroughly, precipitate the protein for 1-3 minutes, and centrifuge at 4°C. S3. Pipette the supernatant into a clean centrifuge tube b, add 4.2 times the volume of cold acetonitrile, mix thoroughly, and place in a -20°C refrigerator for protein precipitation; S4. Remove the protein precipitate sample from the -20°C freezer and centrifuge at 4°C, discard the supernatant; add an appropriate amount of cold acetonitrile to the precipitate, mix thoroughly, wash the precipitate and the wall of the centrifuge tube, centrifuge at 4°C, discard the supernatant, and air dry; S5. Add 0.1% formic acid aqueous solution, vortex and shake to fully dissolve to obtain the test sample; The precipitant is mixed in a volume ratio of acetic acid: acetonitrile: acetone = 1:4:15; The chromatographic conditions for the detection are as follows: Chromatographic column: Phenomenex Kinetex C8 column, 50 x 2.1 mm, 2.6 μm, 100 A; Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile; Flow rate: 0.4 mL / min; Column temperature: 55°C; Injection volume: 15 µL The gradient elution method is as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; the gradient elution time is 4.5 minutes and then stops, where: 0.01-0.5 min: the volume fraction of mobile phase A was 95%; The volume fraction of mobile phase A decreased from 95% to 80% at 0.5-0.6 min; The volume fraction of mobile phase A decreased from 80% to 50% during 0.6-2 min; 2-2.1 min: the volume fraction of mobile phase A decreased from 50% to 0; 2.1-3.1 min: the volume fraction of mobile phase A was kept at 0; 3.1-3.2 min The volume fraction of mobile phase A decreased from 0 to 95%; The volume fraction of mobile phase A was maintained at 95% from 3.2 to 4.5 min.

2. The method for detecting insulin-like growth factor in blood based on HPLC-MS / MS according to claim 1, characterized in that: The blank matrix was 2% BSA solution.

3. The method for detecting insulin-like growth factor in blood based on HPLC-MS / MS according to claim 1, characterized in that: The mass spectrometry conditions for the detection are as follows: Ion source: electrospray ionization source, positive ion mode; capillary voltage: 5500 V; ion source temperature: 500 °C; ion source nebulizer gas: 55 psi; ion source heating auxiliary gas: 55 psi; Curtain gas: 40 psi; Collision gas: Medium; Scan mode: MRM, MRM parameters: Quantitative ion: IGF-1_8_y5; Q1: 956.950 m / z; Q3: 473.500 m / z; RT: 1.65 min; DP: 100 V; EP: 10V; CE: 45V; CXP: 15V.

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