Kit for simultaneously determining IGF-1 and IGF-2 in serum based on liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a kit for simultaneously determining IGF-1 (Insulin-like Growth Factor) and IGF-2 (Insulin-like Growth Factor-2) in serum on the basis of liquid chromatography-tandem mass spectrometry. The kit can be used for simultaneously determining the IGF-1 and the IGF-2 in the serum. During use, after pretreatment, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) is adopted for analysis. According to the present invention, the method has the following characteristics that the linearity of the IGF-1 is good in the range of 18.16 ng / mL to 1000 ng / mL, the linearity of the IGF-2 is good in the range of 23.71 ng / mL to 1500 ng / mL, and the method can be used for simultaneously determining the IGF-1 and the IGF-2 in the serum. The method is simple, convenient, rapid, accurate, reliable and good in repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental safety detection, and in particular to a kit for simultaneously determining serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry. Background Art

[0002] Insulin-like growth factor 1 and insulin-like growth factor 2 (IGF-1 and IGF-2) are widely used biomarkers, mainly for auxiliary diagnosis and treatment monitoring of growth hormone (GH)-related diseases, such as acromegaly, gigantism and dwarfism (laron type).

[0003] The quantitative detection of IGF-1 and IGF-2 is traditionally performed using ligand binding assays on immunoassay platforms. Since traditional methods rely on immunoreagents (such as antibodies, etc.), their quantitative repeatability is poor and they are easily affected by endogenous interference. At the same time, the antibody dependence also leads to poor consistency between laboratories, and the reference values ​​in different regions or laboratories show significant differences. It is precisely because of the use of different antibodies, calibration, extraction methods, and even different international reference standards between kits that traditional immunoassay methods lack consistency on different platforms, which makes it difficult to formulate relevant laboratory testing guidelines. Although clinical attempts to establish unified testing standards have been made, the results are minimal. Therefore, improving or even developing new methods for detection has important prospects and value in clinical practice and research. In summary, the technical difficulties of simultaneous detection of IGF-1 and IGF-2 in the existing technology are mainly manifested in the following aspects: (1) poor quantitative repeatability (2) easy to be affected by endogenous interference (3) low sensitivity.

[0004] Therefore, there is an urgent need to develop a kit that can avoid cross infection and measure serum IGF-1 and IGF-2 with high sensitivity. Summary of the invention

[0005] The main purpose of the present invention is to provide a kit for simultaneously determining serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry, which avoids cross infection and has high sensitivity.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The present invention provides a kit for simultaneously determining serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry, the kit comprising the following reagents:

[0008] a) Standard curve stock solution: rabbit serum matrix solution containing 50 ng / μL IGF-1 and 50 ng / μL IGF-2;

[0009] b) Internal standard storage solution: 10 μg / mL 15N-labeled IGF-1 solution;

[0010] c) Pretreatment reagents: 0.8%-1.2% acetic acid aqueous solution and 99.7%-100% acetonitrile.

[0011] Furthermore, the rabbit serum is a special matrix for detecting human IGF.

[0012] Furthermore, the acetic acid aqueous solution and acetonitrile are used in the pretreatment step, the volume ratio of the acetic acid aqueous solution with a mass fraction of 0.8%-1.2% to the sample is 2:1, and the volume ratio of the acetonitrile to the sample is 4:1.

[0013] Furthermore, the kit also includes: an internal standard 15 N-IGF-1.

[0014] The present invention also provides a method for simultaneously determining serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry, wherein the method adopts ultra-high performance liquid chromatography-tandem mass spectrometry UPLC-MS / MS for analysis after pretreatment, and the method comprises the following steps:

[0015] (1) Preparation of standard solution and standard curve;

[0016] (2) Sample pretreatment;

[0017] (3) High performance liquid chromatography-tandem mass spectrometry analysis; the high performance liquid chromatography-tandem mass spectrometry method includes using a chromatographic column: Acquity BEH C18, 130A, 1.7 μm, 2.1x100 mm.

[0018] Preferably, in the sample pretreatment, 100ul of sample needs to firstly add 2 volumes (200ul) of 1% acetic acid (dissociation solution) and then add 4 volumes (400ul) of acetonitrile (precipitant).

[0019] Preferably, the HPLC-MS / MS method comprises a column flow rate of 0.3 mL / min; an injection volume of 10 μL; a mobile phase of 0.1% formic acid (FA) as phase A and 99.7%-100% acetonitrile (ACN) as phase B.

[0020] Preferably, the HPLC-MS / MS gradient elution program is as follows: 0-1 min, 10% B phase; 1-10.5 min, 60% B phase; 10.5-12.5 min, 90% B phase; 12.5-12.6 min, 10% B phase; 12.6-14.7 min, 10% B phase.

[0021] Preferably, the high performance liquid chromatography-tandem mass spectrometry method includes mass spectrometry conditions of electrospray ionization source positive ion mode (ESI+); capillary voltage: 4kV, warping gas / auxiliary gas: GS1:40GS2:10, ion source temperature: 300 degrees.

[0022] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0023] The present invention provides a kit for simultaneously determining serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry, including specific matrix selection, internal standard application and pre-treatment solution setting. These combinations bring unexpected technical effects, such as improving recovery rate, reducing interference, reducing costs, etc., which are specifically manifested in:

[0024] (1) The present invention is a rapid and simultaneous quantitative method for serum IGF-1 and IGF-2. The method is simple to operate, has good repeatability and high sensitivity: the detection sensitivity is 0.1 ng / mL, and the intra-batch CV is less than 5%. The pretreatment time is shortened to 30 minutes, the recovery rate is increased by 20%, and the cost of a single test is reduced by 40%.

[0025] (2) The present invention uses rabbit serum matrix standard: Because rabbit IGF-1 / 2 and human IGF-1 / 2 protein sequences are different, they will not cause interference in mass spectrometry detection. Therefore, rabbit serum is used as the matrix to prepare IGF-1 and IGF-2 standard curves and detect the concentration of human serum IGF-1 / 2, thereby eliminating cross-interference during mass spectrometry detection and meeting the matrix matching requirements.

[0026] (3) The present invention adopts the acid hydrolysis-organic precipitation combined technology: 0.8%-1.2% acetic acid dissociates the IGF-binding protein complex, and the synergistic effect of acetonitrile precipitation of impurities is combined to achieve the best target peak intensity effect, and no flocculent precipitate is produced by naked eye, thereby improving the recovery rate of the target (>95%).

[0027] (4) The present invention uses an internal standard (15N-labeled IGF-1) as an internal reference for both IGF-1 and IGF-2 to correct the error in the experiment and make the quantitative analysis more accurate. Universal stable isotope internal standard: 15 N-labeled IGF-1 can simultaneously correct the difference in ionization efficiency between IGF-1 and IGF-2 and reduce the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 IGF-1, 15 Chromatograms of N-IGF-1 and IGF-2;

[0029] Figure 2 is the standard curve of IGF-1 and IGF-2;

[0030] Figure 3 Linear regression equation and percentage of difference for methodological comparison between chemiluminescence and ultra-performance liquid chromatography-tandem mass spectrometry for IGF-1;

[0031] Figure 4 is the concentration distribution of IGF-1 and IGF-2 in the normal population, where A is the concentration distribution of IGF-1 in the normal population by age (year), B is the concentration distribution of IGF-1 in the normal population by age (month), C is the concentration distribution of IGF-1 in the normal population by gender, D is the concentration distribution of IGF-2 in the normal population by age (year), E is the concentration distribution of IGF-2 in the normal population by age (month), and F is the concentration distribution of IGF-2 in the normal population by gender.

[0032] Figure 5 The LOQ and linearity verification and carryover contamination assessment data of IGF-1 and IGF-2. A is the LOQ test concentration and corresponding imprecision of IGF-1 and IGF-2, B is the LOQ experimental concentration and imprecision fitting curve of IGF-1, C is the LOQ experimental concentration and imprecision fitting curve of IGF-2, D is the linearity verification concentration ratio and test results of IGF-1 and IGF-2, L% refers to the corresponding low value concentration ratio, H% refers to the high value concentration ratio, E is the linearity verification fitting curve of IGF-1, F is the linearity verification fitting curve of IGF-2, and G is the sample concentration of IGF-1 and IGF-2 mass spectrometry carryover contamination assessment test;

[0033] Figure 6 Intra-assay and inter-assay precision and accuracy data for IGF-1 and IGF-2;

[0034] Figure 7 The results are shown in Figure 2. The results are shown in Figure 2. The results are shown in Figure 2. The results are shown in Figure 2. The results are shown in Figure 2. The results are shown in Figure 2. The results are shown in Figure 2. The results are shown in Figure 2.

[0035] Figure 8 The results are shown in Figure 1. The results are shown in Figure 2. The results are shown in Figure 3. The results are shown in Figure 4. The results are shown in Figure 5. IGF-1 and IGF-2 were incubated with 99.9% acetonitrile and then subjected to acid hydrolysis and organic precipitation after incubation with 0.8% AA, 1% AA, and 1.2% AA, respectively. DETAILED DESCRIPTION

[0036] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0037] Example 1. Kit and method for simultaneous determination of serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry. 1. Kit composition:

[0038] (1) Standard curve working solution: IGF-1 / 2 gradient solution prepared with rabbit serum (0.5-200 ng / mL);

[0039] (2) Internal standard working solution: 15 N-IGF-1 (100 ng / mL);

[0040] (3) Pretreatment reagents: 1% acetic acid aqueous solution, 99.9% acetonitrile precipitant;

[0041] 2. Methods

[0042] 1. Reagent preparation

[0043] (1) Reagents

[0044] IGF-1, lyophilized powder;

[0045] 15 N-labeled IGF-1, lyophilized powder;

[0046] IGF-2, lyophilized powder;

[0047] rabbit serum;

[0048] (2) Consumables

[0049] LoBind centrifuge tubes, pipette tips;

[0050] (3) Reagent pretreatment

[0051] Prepare 10mM acetic acid: add 6ul of pure acetic acid to 10ml of pure water and mix well for later use;

[0052] Centrifugation: Centrifuge at 10,000 rpm for 30 seconds. The lyophilized powder will adhere to the tube wall or tube cap, and centrifugation will collect the lyophilized powder at the bottom of the tube;

[0053] IGF-1 / IGF-2: Add 10 mM acetic acid / water, mix gently with a pipette tip, and resuspend to a concentration of 100 μg / mL;

[0054] 15 N-labeled IGF-1: Add 10 mM acetic acid / water, mix gently with a pipette tip, resuspend to a concentration of 10 μg / mL, dispense 100 ul into each tube, and freeze at -80 degrees;

[0055] (4) Pretreatment Note:

[0056] Reconstitution: Add reconstitution buffer / sterile water, mix gently with a pipette tip, and resuspend to a concentration of not less than 100 μg / mL.

[0057] Storage: If the experimental period is less than 1 week, it can be stored at 2-8℃. If the experimental period is greater than 1 week, dilute with a solution containing carrier protein (0.1% BSA, 5% HSA, 10% FBS, 5% trehalose), aliquot and freeze. Long-term storage at -80℃ is recommended.

[0058] Notes: The carrier protein can pre-block the protein binding sites on the tube wall so that it will not adhere to the tube wall. Before aliquoting and freezing, diluting with a solution containing the carrier protein can ensure that low-concentration recombinant proteins still maintain a high stability.

[0059] (5) Reagent stock solution:

[0060] Prepare 10 ng / uL IGF-1: Add 100 ul of IGF-1 (100 ng / uL) to a LoBind centrifuge tube, then add 900 ul of blank rabbit serum, vortex for more than 10 seconds to mix thoroughly, dispense 60 ul into each tube, and freeze at -80 degrees;

[0061] Prepare 10 ng / uL IGF-2: Add 100 ul of IGF-2 (100 ng / uL) to a LoBind centrifuge tube, then add 900 ul of blank rabbit serum, vortex for more than 10 seconds to mix thoroughly, dispense 50 ul into each tube, and freeze at -80 degrees;

[0062] (6) Standard curve

[0063] Prepare the standard curve according to the following ratio:

[0064] Table 1

[0065] level Punctuation, ng / mL Add 50ng / uL IGF-1 / IGF-2 Rabbit serum,ul 6 1000IGF-1; 1500IGF-2; 10ul IGF-1; 15ul IGF-2; 475 5 600IGF-1; 900IGF-2; 300ul level 6 200 4 400IGF-1; 600IGF-2; 300ul level 5 150 3 200IGF-1; 300IGF-2; 200ul level 4 200 2 100IGF-1; 150IGF-2; 200ul Level 3 200 1 50IGF-1; 75IGF-2; 200ul Level 2 200 0 0 0ul 100

[0066] 2. Test Procedure Experimental Steps

[0067] (1) Prepare 1% acetic acid: add 100 μl of pure acetic acid to 9.9 mL of pure water and mix well for later use;

[0068] (2) Accurately pipette 100ul of the sample to be tested / quality control, add 60ng (6μL) of 15 N-IGF-1, then add 200 μL of 1% acetic acid, vortex for 5 min to mix thoroughly;

[0069] (3) Add 400 μl of acetonitrile and vortex gently for more than 10 min at room temperature;

[0070] (4) Centrifuge at 18,000 g for 10 min at 4°C, take 630 μl of supernatant, and drain at 40-45°C for 105 min;

[0071] (5) Add 50 μL of 1% acetic acid to reconstitute the sample, gently vortex at room temperature for more than 5 min, centrifuge at 15000 g, 4 °C, 5 min, and analyze on a centrifuge;

[0072] 3. High Performance Liquid Chromatography and Mass Spectrometry Tandem Analysis

[0073] 1. Chromatographic parameters (Thermo UltiMate 3000UHPLC)

[0074] Column: Acquity BEH C18, 130A, 1.7 μm, 2.1 x 100 mm

[0075] Phase A: 0.1% FA

[0076] Phase B: ACN

[0077] gradient:

[0078] Table 2

[0079] 0min 10％B 1min 10％B 10.5min 60％B 10.6min 90％B 12.5min 90％B 12.6min 10％B 14.7 10％B

[0080] Flow rate: 0.3mL / min

[0081] Column temperature: 50 degrees

[0082] Injection volume: 10ul

[0083] IGF-1 retention time: 9.10min

[0084] IGF-2 retention time: 8.78min

[0085] 2. Mass spectrometry parameters (Thermo Scientific Q-Exactive)

[0086] ESI, positive ion mode

[0087] Capillary voltage: 4kV

[0088] Warping gas / auxiliary gas: GS1:40GS2:10

[0089] Ion source temperature: 300 degrees

[0090] Ion Pair:

[0091] Table 3

[0092] 1106.47329 15N-IGF1,7+ 968.42114 15N-IGF1,8+ 860.93079 15N-IGF1,9+ 1093.51807 IGF1,7+ 956.95538 IGF1,8+ 850.73914 IGF1,9+ 1067.93848 IGF2,7+ 934.57202 IGF2,8+ 830.84320 IGF2,9+

[0093] IGF-1, 15 The chromatograms of N-IGF-1 and IGF-2 are shown in Figure 1 shown.

[0094] Experimental Example 1: Effect Testing

[0095] 1. Linear range and detection limit

[0096] UPLC-MS / MS analysis was performed using a mixed standard working solution of 0-1000 ng / mL (IGF-1) / 0 and 0-1500 ng / mL (IGF-2). The standard curve was drawn with the injected mass concentration as the abscissa (x) and the quantitative ion pair peak area as the ordinate (y). The standard curves of IGF-1 and IGF-2 are shown in Figure 1. Figure 2 The results showed that the standard solution had a good linear relationship in the range of 0-1000 ng / mL (IGF-1) and 0-1500 ng / mL (IGF-2).

[0097] The conventional IGF-1 chemiluminescence method of the prior art and the ultra-high performance liquid chromatography-tandem mass spectrometry method of the present application were compared by linear regression equation and difference percentage. The results are as follows: Figure 3 As shown, Figure 3 The results showed that the ultra-high performance liquid chromatography-tandem mass spectrometry and chemiluminescence methods were highly correlated and comparable for IGF-1. Since there is currently no chemiluminescence detection kit for IGF-2, no comparison was performed.

[0098] The sample with known concentration was diluted proportionally, and the diluted sample was tested repeatedly and the coefficient of variation CV value was calculated. The fitting curve of sample concentration and CV was drawn, and the corresponding concentration value of the maximum acceptable coefficient of variation was calculated to determine the limit of quantitation (LOQ) value. Figure 5 As shown in A and B, the LOQs of IGF-1 and IGF-2 were 18.16 ng / mL and 23.71 ng / mL, respectively.

[0099] 2. Spiked recovery and precision

[0100] Precision experiments and recovery tests were performed on samples of certain concentrations using optimized conditions. The spike levels were 200 ng / mL for AD1 and 400 ng / mL for AD2, 5 times a day for 5 consecutive days.

[0101] The results are as follows Figure 6 As shown, the intra-batch precision of the three concentrations of IGF-1 was 4.25%-12.24%, the inter-batch precision was 11.40%-14.78%, and the recoveries were 86.91%-118.73% when the spiked concentrations were 200ng / mL and 400ng / mL; the intra-batch precision of the three concentrations of IGF-2 was 3.22%-12.85%, the inter-batch precision was 6.93%-12.27%, and the recoveries were 95.50%-116.83% when the spiked concentrations were 200ng / mL and 400ng / mL, indicating that the method met the determination requirements and had good precision and accuracy.

[0102] 3. Linearity evaluation and carryover evaluation of actual samples

[0103] Fresh samples were used to evaluate the linearity of the established methodology. One low-concentration (L) and one high-concentration (H) fresh plasma specimen were selected. The low-concentration (L) and high-concentration (H) were diluted in a series of proportions. The concentrations covered the linear range provided in the reagent instructions. Each sample was tested twice. The mean and expected value of the test results were subjected to multivariate regression statistics. The judgment criteria were calculated based on EP6-A2 as the judgment criteria.

[0104] Test results such as Figure 5 As shown in D, E, and F: The linear validation equations for IGF-1 and IGF-2 are linear equations. The established methodology was evaluated for carryover contamination. Negative samples and high-value samples were continuously monitored in a certain order, and the carryover contamination rate was calculated as follows: Figure 5 As shown in G, there is no carryover contamination between samples in the detection of IGF-1 and IGF-2 by this method, indicating that the kit provided by the present invention has good results for the assessment of carryover contamination and meets the measurement requirements.

[0105] 4. Normal population reference area

[0106] The method established by this kit was used to analyze a certain number of samples from children and adults in an apparently healthy population, such as Figure 4 As shown in Table 4, the following results are obtained.

[0107] Table 4

[0108]

[0109]

[0110] Depend on Figure 4 From the data in Table 4, we can see that:

[0111] (1) Relationship between IGF-1 levels and age

[0112] Trend: IGF-1 levels show a clear trend of first increasing and then decreasing with age.

[0113] Childhood (<1 year to 10 to 20 years old): It rises rapidly from 26.26 ng / mL (<1 year old) to a peak of 295.45 ng / mL (10 to 20 years old), which is consistent with the physiological characteristics of vigorous secretion of growth hormone during adolescence.

[0114] Adulthood (after 20 years old): gradually decreases from 243.12 ng / mL in 20-30 years old to 156.33 ng / mL in >50 years old, which may be related to the decrease in growth hormone secretion.

[0115] Statistical significance: p<0.001, indicating that the difference in IGF-1 levels between different age groups is highly statistically significant.

[0116] (2) Relationship between IGF-2 levels and age

[0117] Trend: IGF-2 levels fluctuate and decrease with age, but the trend is not as clear as that of IGF-1.

[0118] Infancy (<1-5 years old): decreased from 762.58 ng / mL (<1 year old) to 728.66 ng / mL (1-5 years old), which may be related to the gradual adjustment after high expression in the fetal period.

[0119] Adulthood (after 20 years old): large fluctuations (e.g., the 30-50 age group rebounds to 339.48 ng / mL), but generally lower than childhood.

[0120] Statistical significance: p = 0.042, indicating that the difference between age groups is statistically significant, but the significance level is low, which may be affected by data variation or sample size.

[0121] (3) In summary,

[0122] The data measured by the method of the present invention support the conclusion that "IGF-1 levels reach a peak during puberty and then gradually decrease with age", which is consistent with known physiological laws (such as growth hormone dependence). p<0.001 shows that age is an important influencing factor of IGF-1 levels. This is the same as the conclusion known in the prior art, indicating that the method of the present invention has practical application prospects.

[0123] Example 2

[0124] The pre-treatment agent of this embodiment is 0.8% acetic acid aqueous solution + 99.9% acetonitrile, and other conditions are the same as those of Example 1.

[0125] Example 3

[0126] The pre-treatment agent of this embodiment is 1.2% acetic acid aqueous solution + 99.9% acetonitrile, and other conditions are the same as those of embodiment 1.

[0127] Comparative Example 1

[0128] The pre-treatment agent of this comparative example is 2% acetic acid aqueous solution + 99.9% acetonitrile, and other conditions are the same as those of Example 1.

[0129] Comparative Example 2

[0130] The pre-treatment agent of this comparative example is 5% acetic acid aqueous solution + 99.9% acetonitrile, and other conditions are the same as those of Example 1.

[0131] Experimental Example 2: Comparison of the effects of pretreatment agents

[0132] The acid hydrolysis-organic precipitation combination after incubation of IGF-1 and IGF-2 in Example 1, Example 2, Example 3 and Comparative Examples 1 and 2 was measured and statistically analyzed.

[0133] Table 5

[0134]

[0135] Depend on Figure 7 From Table 5, we can see that:

[0136] In Example 1, 1% AA + 99.9% acetonitrile was used for incubation, and the target peak intensity effect was the best.

[0137] In Comparative Example 1, when incubated with 2% AA, the intensity of the target peak decreased slightly.

[0138] In Comparative Example 2, when incubated with 5% AA, the intensity of the target peak decreased significantly and the number of interfering peaks increased.

[0139] Depend on Figure 8 From Table 5, we can see that:

[0140] The peak intensities and peak shapes of Example 2 (0.8% AA + 99.9% acetonitrile), Example 1 (1% AA + 99.9% acetonitrile), and Example 3 (1.2% AA + 99.9% acetonitrile) are similar, and there is no obvious interfering peak.

[0141] In summary, the acid hydrolysis-organic precipitation combination after incubation of IGF-1 and IGF-2 in the concentration range of 0.8% to 1.2% AA + 99.9% acetonitrile is better than other concentrations, and 0.8% to 1.2% AA + 99.9% acetonitrile produces an unexpected synergistic effect.

[0142] The above implementation modes are merely descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A kit for simultaneously determining serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry, characterized in that: The kit includes the following reagents: a) Standard curve stock solution: rabbit serum matrix solution containing 50 ng / μL IGF-1 and 50 ng / μL IGF-2; b) Internal standard storage solution: 10 μg / mL 15 N-labeled IGF-1 solution; c) Pretreatment reagents: 0.8%-1.2% acetic acid aqueous solution and 99.7%-100% acetonitrile.

2. A kit for simultaneously determining serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The rabbit serum is a special matrix for detecting human IGF.

3. A kit for simultaneously determining serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The acetic acid aqueous solution and acetonitrile are used in the pretreatment step, the volume ratio of the acetic acid aqueous solution with a mass fraction of 0.8%-1.2% to the sample is 2:1, and the volume ratio of the acetonitrile to the sample is 4:

1.

4. A kit for simultaneously determining serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The kit also includes: an internal standard 15 N-labeled IGF-1.

5. A kit for simultaneously determining serum IGF-1 and IGF-2 based on liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The kit also includes: 0.1% formic acid and 99.7%-100% acetonitrile.

6. Use of the reagents in the kit according to any one of claims 1 to 5 in the preparation of a product for determining serum IGF-1 and IGF-2.

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