QAMS sampling-based insulin-like growth factor detection method and kit

Abstract

The invention provides an insulin-like growth factor detection method and kit based on QAMS sampling, and belongs to the technical field of protein biological detection.The insulin-like growth factor detection method comprises the steps that a quantitative absorption microsampler is used for conducting peripheral blood sample collection on a sampling object, and an equivalent dried blood spot sample is obtained; the method comprises the following steps: extracting a supernatant sample containing an insulin-like growth factor internal standard working solution through pretreatment: transferring a dried blood spot sample into a centrifugal tube or a pore plate, adding the prepared insulin-like growth factor internal standard working solution, carrying out ultrasonic treatment, adding an acetonitrile solution containing acetic acid to remove protein, carrying out normal-temperature vortex incubation, adding an ammonia water solution, and carrying out ultrasonic treatment to obtain a supernatant sample containing the insulin-like growth factor internal standard working solution; centrifuging to obtain a supernate sample, and carrying out SPE (Solid Phase Extraction) purification on the extracted supernate sample; and detecting the content of the insulin-like growth factor in the extracted and SPE-purified supernate sample by adopting liquid chromatography-mass spectrometry to obtain the concentration of the insulin-like growth factor. The detection method disclosed by the invention has the advantage of ensuring that the IGF-1 detection concentration is close to the real concentration of a sampled human body.

Description

Technical Field

[0001] The present invention relates to the technical field of protein biological detection, and in particular to an insulin-like growth factor detection method and a kit based on QAMS sampling. Background Art

[0002] Insulin-like growth factors (IGFs) are a class of multifunctional cell proliferation regulatory factors. They are polypeptide protein substances that are similar to insulin in molecular structure, also known as insulin-like growth factors. They are composed of two types of substances, namely IGF-I and IGF-II. In the human body, IGF-I is the main component of insulin-like growth factors. In addition to liver cells, which are the main secretory cells, up to a dozen cells such as kidney cells and spleen cells can secrete IGF-I. IGF-I is affected by genetics, hormone levels, nutritional status and other factors in the human body. It has extremely important and rich biological functions, including promoting cell proliferation and differentiation, promoting growth, inhibiting cell apoptosis, etc. It plays an important role in the pathophysiological processes of cardiovascular diseases, endocrine metabolic diseases and tumors. IGF-I is composed of 70 amino acids and is a heat-resistant alkaline single-chain polypeptide with endocrine, autocrine and paracrine properties. The molecular weight is 7649Da.

[0003] There are currently a variety of IGF-1 detection methods on the market, including immunoassays, blood biochemical tests, etc. Among them, chemiluminescence immunoassay (CLIA) is currently the more commonly used method for detecting IGF-1. Mass spectrometry (MS) is a detection method that has been widely used in clinical practice in recent years. It has the advantages of high specificity and the ability to detect multiple proteins at the same time.

[0004] Well-preserved blood samples play a decisive role in the test results of IGF-1. However, existing IGF-1 detection technology focuses more on the laboratory stage and ignores the collection and preservation of blood samples. Since IGF-1 in the blood is easily affected by factors such as temperature, light, and pH value and degrades or denatures, it is easy to cause the test results to be inconsistent with the actual results, and the IGF-1 concentration in the human body cannot be accurately determined. Summary of the invention In response to the above problems, the present invention provides an insulin-like growth factor detection method and kit based on QAMS sampling, which reduces the impact of the storage environment on the blood sample, thereby accurately and quantitatively detecting the concentration of insulin-like growth factor in the blood sample, so as to reflect the actual IGF-1 concentration in the human body of the sampled subject to the greatest extent.

[0005] The present invention provides a method for detecting insulin-like growth factor based on QAMS sampling, comprising: The peripheral blood samples of the sampling object are collected by using a quantitative absorption micro-sampler with integrated collection and storage to obtain 10uL-20uL equivalent dry blood spot samples; Pre-treating the dried blood spot sample, extracting a supernatant sample containing an insulin-like growth factor internal standard working solution, and releasing the insulin-like growth factor without using a surfactant, including transferring the dried blood spot sample to a centrifuge tube or a well plate and adding the prepared insulin-like growth factor internal standard working solution, adding an acetic acid-containing acetonitrile solution to remove protein after ultrasonication or oscillation, incubating at room temperature with vortexing and adding an ammonia solution to adjust the pH, obtaining a supernatant sample by centrifugation, and performing SPE purification on the extracted supernatant sample; Liquid chromatography-mass spectrometry is used to detect the insulin-like growth factor content of the supernatant sample extracted and SPE purified to obtain the insulin-like growth factor concentration, including configuring liquid chromatography-mass spectrometry, preparing a calibration curve and detecting the supernatant sample after SPE purification based on the calibration curve to obtain the content of insulin-like growth factor, wherein the mobile phase A and the mobile phase B of the chromatography are formic acid water and pure acetonitrile respectively, and gradient elution is used for separation.

[0006] As a further improvement of the present invention, the internal standard working solution contains 0.1% BSA.

[0007] As a further improvement of the present invention, the SPE purification includes activating and balancing the SPE column with methanol and ammonia solution, followed by secondary elution and secondary washing respectively; wherein the first elution uses a 5% ammonia solution, the second elution uses a 5% methanol solution containing 1% acetic acid, the first elution uses a methanol: water: acetic acid mixed solution, and the second elution uses pure water.

[0008] As a further improvement of the present invention, the elution gradients of the mobile phase A and the mobile phase B are:

[0009] Wherein, the mobile phase A is 0.1% formic acid water.

[0010] As a further improvement of the present invention, the ESI source parameters of the mass spectrometer include a capillary voltage of 1.0 kV, a desolvation gas temperature of 600° C., and a desolvation gas flow rate of 1100 L / hr.

[0011] As a further improvement of the present invention, the preparation of the calibration curve includes preparing a high-concentration mother solution, and diluting it with a mother solution diluent and a first matrix diluent to obtain a plurality of calibration products, and detecting the plurality of calibration products to obtain a calibration curve, wherein the mother solution is 10% acetic acid, 30% methanol, and 60% water, and the first matrix diluent is 5% BSA+0.1% Proclin300 preservative.

[0012] As a further improvement of the present invention, the quantitative absorption micro-sampler contains a deoxidizing desiccant.

[0013] As a further improvement of the present invention, the ultrasonic or oscillation time is not less than 30 minutes.

[0014] As a further improvement of the present invention, the vortex speed of the room temperature vortex incubation is 900 rpm, and the vortex time is 10 min.

[0015] As a further improvement of the present invention, the centrifugation time is 10 min and the centrifugal force is 14000×g.

[0016] The invention provides an insulin-like growth factor detection kit based on QAMS sampling, comprising an internal standard working solution, a protein precipitant, an activation solution, a balancing solution, a washing solution and an eluent.

[0017] The present invention provides an application of an insulin-like growth factor detection kit based on QAMS sampling in detecting insulin-like growth factor.

[0018] The present invention provides an insulin-like growth factor detection method and kit based on QAMS sampling, wherein a peripheral blood sample of a sampling object is collected by using a quantitative absorption microsampler with integrated collection and storage to obtain a 10uL-20uL equivalent dry blood spot sample; the dry blood spot sample is pre-treated to extract a supernatant sample containing an insulin-like growth factor internal standard working solution, and the insulin-like growth factor is released without using a surfactant to dissociate, including transferring the dry blood spot sample to a centrifuge tube or a well plate and adding the prepared insulin-like growth factor internal standard working solution, adding an acetic acid-containing acetonitrile solution to remove protein after ultrasound or vibration, vortexing and incubating at room temperature and adding an ammonia solution to adjust the pH, obtaining a supernatant sample by centrifugation, and performing SPE purification on the extracted supernatant sample. chemistry; using liquid chromatography-mass spectrometry to detect the insulin-like growth factor content of the supernatant sample extracted and purified by SPE to obtain the insulin-like growth factor concentration, including configuring liquid chromatography-mass spectrometry, preparing a calibration curve and detecting the supernatant sample after SPE purification based on the calibration curve to obtain the content of insulin-like growth factor, wherein the mobile phase A and the mobile phase B of the chromatography are formic acid water and pure acetonitrile respectively, and gradient elution is used for separation, thereby ensuring the stability of the test sample, ensuring that the IGF-1 detection concentration is close to the actual concentration of the sampled human body, accurately determining the IGF-1 concentration in the human body, and the required detection limit is low, only a trace amount of blood sample needs to be collected and dried to form a dry blood spot sample, which greatly reduces the amount of blood collected in the existing detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1It is a flow chart of an insulin-like growth factor detection method based on QAMS sampling according to an embodiment of the present invention.

[0020] Figure 2 It is a structural schematic diagram of a QAMS device in the insulin-like growth factor detection method using QAMS sampling according to an embodiment of the present invention.

[0021] Figure 3 This is a graph showing the changes of mobile phase A and mobile phase B over time in the method for detecting insulin-like growth factor based on QAMS sampling in an embodiment of the present invention.

[0022] Figure 4 It is a schematic diagram of a calibration curve of the insulin-like growth factor detection method based on QAMS sampling according to an embodiment of the present invention.

[0023] Figure 5 This is an IGF-1 detection spectrum of an embodiment of the present invention.

[0024] Explanation of the reference numerals: 1. cover body; 11. upper cover; 2. box body; 21. bottom box; 22. perspective part; 3. quantitative blood collection tube; 31. first tube body; 32. second tube body; 33. connecting part; 4. modified sample carrier; 5. bracket; 51. support plate; 52. isolation plate; 53. support ring. DETAILED DESCRIPTION

[0025] The following is combined with specific embodiments and appendix Figure 1-5 The invention is described in detail so that those skilled in the art can more fully understand the purpose, features and effects of the invention.

[0026] Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. When the definition of a term in the present invention conflicts with the meaning commonly understood by those skilled in the art to which the present invention belongs, the definition described in the present invention shall prevail.

[0027] The present invention provides an insulin-like growth factor detection method and kit based on QAMS sampling, adopts a quantitative absorption microsampler to collect and store blood samples, thereby ensuring the stability of the samples to be detected, and requires a small amount of sample while ensuring a good detection effect, thereby reducing the amount of blood collected.

[0028] Embodiment 1 As a specific embodiment of the present invention, this embodiment provides an insulin-like growth factor detection method based on QAMS sampling, referring to Figure 1 , the specific steps are as follows: S100, using QAMS to collect blood samples and obtain dried blood spot samples Use Figure 2The QAMS (Quantitative Adsorption MicroSampler) shown in the figure collects peripheral blood samples from the sampling object, such as fingertip blood. Specifically, the QAMS includes a cover body 1 and a box body 2, wherein the cover body 1 includes an upper cover 11 and a structure for mounting and fixing a quantitative blood collection tube 3 located inside the upper cover 11, the cover body 1 and the box body 2 are snap-fitted, and the quantitative blood collection tube 3 extends downward into the box body 2, and the quantitative blood collection tube 3 is a capillary. The quantitative blood collection tube 3 includes a first tube body 31 and a second tube body 32 for collecting blood in different amounts. Both the first tube body 31 and the second tube body 32 can be detachably fixed on the upper cover 11. When it is necessary to collect peripheral blood samples from the sampling object, the first tube body 31 or the second tube body 32 is removed from the upper cover 11. When the blood sample collection is completed, the first tube body 31 or the second tube body 32 is fixed to the upper cover 11 again. The box body 2 includes an upper perspective portion 22 and a lower bottom box 21, and the perspective portion 22 is snap-fitted to the bottom box 21, wherein the perspective portion 22 is snap-fitted to the cover body 1. A bracket 5 for supporting a modified sample carrier 4 is installed at the top of the bottom box 21. The bracket 5 includes a support plate 51 and a support ring 53 located on the top surface of the support plate 51. The number of the support rings 53 is equal to the number of the quantitative blood collection tubes 3 and corresponds one to one. The modified sample carrier 4 is placed on the support ring 53. In addition, the bracket 5 also includes an isolation plate 52 that separates the multiple support rings 53 to reduce the influence between the dry blood spot samples formed on different modified samples 4. The modified sample carrier 4 is used to carry the fresh blood sample in the quantitative blood collection tube 3 and form a carrier for the dry blood spot sample after the fresh blood sample is dried.

[0029] The space inside the bottom box 21 below the bracket 5 is a drying chamber in which a deoxygenating desiccant is placed. The drying chamber is connected to the modified sample carrier 4, thereby accelerating the drying speed of the collected blood sample and maintaining the long-term drying inside the QAMS to ensure the stability of the dried blood spot sample. In addition, a modified sample carrier is used to improve the stability of the test component IGF-1 in the blood sample, reduce the degradation rate of the blood sample, and make the test result closer to the actual situation.

[0030] Specifically, S100 includes: S101. Preparation of consumables, including QAMS, disposable blood collection needle, alcohol cotton and hemostatic cotton; S102. Disinfect the fingertips with alcohol, puncture the fingertips with a disposable blood collection needle, and quickly wipe off the first drop of blood with hemostatic cotton; S103, the capillary of the quantitative blood collection tube 3 of the QAMS quantitatively draws peripheral blood from the fingertips until the capillary is full, avoiding the inhalation of air; S104 , horizontally install the capillary tube for absorbing peripheral blood back into the QAMS, and immediately vertically place the capillary tube on a horizontal table for 2 minutes after the capillary tube is fully in contact with the modified sample carrier 4 , so as to facilitate the transfer of blood to the modified sample carrier 4 .

[0031] After the blood sample is collected and the dried blood spot is formed, it is reserved for testing. Specifically, 20uL of liquid whole blood sample is sucked by the quantitative blood collection tube 3 and then dried to obtain a 20uL liquid blood equivalent sample, or 10uL of liquid whole blood sample is sucked by the quantitative blood collection tube 3 and then dried to obtain a 10uL liquid blood equivalent sample.

[0032] The QAMS blood collection in this step is simpler and can be stored, dried and deoxygenated at the same time, so that the dry blood spot samples obtained after blood collection are maintained in a relatively independent environment, reducing the impact of the external environment on the dry blood spots, ensuring that the dry blood spot samples have the advantages of high stability and non-denaturation, reducing the change in the IGF-1 content in the blood sample, and facilitating subsequent accurate detection.

[0033] QAMS allows blood samples to be quickly separated from the external environment after collection, and reduces the interference of the external environment on the sample during the formation and storage (including transportation) of dried blood spots.

[0034] S200, dried blood spot sample pretreatment S201. Preparation of internal standard working solution The N15 IGF-I internal standard stock solution was diluted with purified water to obtain an internal standard working solution. Specifically, the internal standard original concentration of the N15 IGF-I internal standard stock solution was 2ug / ml, the dilution multiple was 50 times, and the final concentration after dilution was 40ng / ml. The N15 IGF-I internal standard stock solution contained 5% BSA (bovine serum albumin) and contained 0.1% BSA after dilution, thereby reducing container adsorption.

[0035] Optionally, use the standard products provided by ordinary manufacturers to prepare the internal standard working solution, dilute the SRM2926 standard (NIST standard) to a similar concentration, measure under the same conditions, and use the SRM2926 value to calibrate the ordinary manufacturer's value.

[0036] S202, extract supernatant sample The obtained dried blood spot sample is transferred to a centrifuge tube and 400uL of the prepared internal standard working solution is added, and then the sample is treated with an ultrasonic processor. Preferably, the ultrasonic time is not less than 30min, such as 30min-35min. In other embodiments, the obtained dried blood spot sample can also be transferred to a 2mL well plate and 300uL of the prepared internal standard working solution is added, and then the sample is treated with a plate oscillator. Similarly, the oscillation time is not less than 30min, such as 30min-35min. Ultrasound or oscillation of less than 30min can ensure the full and thorough extraction of the IGF-1 component in the dried blood spot sample.

[0037] The dried blood spot sample is a sample containing 20uL of liquid blood equivalent or 10uL of liquid blood equivalent. Preferably, the dried blood spot sample is a sample containing 10uL of liquid blood equivalent. The detection method of this embodiment can realize subsequent detection by using 20uL or even lower 10mL liquid blood equivalent sample, thereby greatly reducing the amount of blood collected, which can greatly reduce the pain and discomfort of the sampled object, especially for children, the elderly and the weak and sick groups. This improvement is particularly important; in addition, it also significantly improves the collection efficiency.

[0038] After sonication was completed using an ultrasonic processor, 200 uL of acetonitrile solution containing 5% acetic acid was added to the dried blood spot sample for protein removal.

[0039] In other embodiments, when a 2 mL well plate is used, preferably, 150 uL of acetonitrile solution containing 5% acetic acid is added to the dried blood spot sample for protein removal.

[0040] Afterwards, the sample is vortexed and incubated at room temperature. Preferably, the vortex speed is 900 rpm and the vortex time is 10 min. Meanwhile, 400 uL of 5% ammonia solution is added and the sample is briefly vortexed to adjust the pH.

[0041] In other embodiments, when a 2 mL well plate is used, preferably, 300 uL of 5% ammonia solution is added and the sample is briefly vortexed to adjust the pH.

[0042] Finally, the supernatant sample is extracted by centrifugation, preferably at 14,000 × g for 10 min.

[0043] S203, SPE purification The extracted supernatant sample mixed with the internal standard working solution was further processed and purified by SPE (Solid-Phase Extraction).

[0044] First, the SPE column is activated and equilibrated using methanol and ammonia solution. Preferably, the activation uses 200uL of methanol and the equilibration uses 200uL of 5% ammonia solution.

[0045] The entire supernatant sample is loaded onto the SPE column, followed by elution and washing, wherein the elution and washing are each divided into two steps. It should be noted that the SPE column capacity limit must be divided into two steps when loading, each time not exceeding 600 uL. In other embodiments, when a 2 mL well plate is used, 700 uL of the extracted supernatant sample is loaded.

[0046] Specifically, the first elution uses an ammonia solution, and the second elution uses a methanol solution. Preferably, the first elution uses 200uL of 5% ammonia solution, and the second elution uses 200uL of 5% methanol solution containing 1% acetic acid.

[0047] The first elution uses a methanol: water: acetic acid mixed solution, and the second elution uses pure water. Preferably, the first elution uses 2x25 uL 60:30:10 methanol: water: acetic acid mixed solution, and the second elution uses 50uL pure water. In other embodiments, the first elution uses 2x20 uL 70:30:10 methanol: water: acetic acid mixed solution, and the second elution uses 40uL pure water.

[0048] The detection method of this embodiment does not need to use surfactants such as CHAPS (3-[(3-cholamidopropyl) dimethylammonium]-1-propanesulfonate), TFE (trifluoroethanol) or SDS (sodium dodecyl sulfate) in pretreatment to dissociate and release IGF-1, thereby reducing the risk of subsequent mass spectrometry contamination.

[0049] S300, detection of insulin-like growth factor content by liquid chromatography-mass spectrometry S301, configure liquid chromatography-mass spectrometry (LC / MS) parameters The chromatographic column used was Peptide C18 1.8um, 50mm.

[0050] The injection volume is 10 uL. In other embodiments, when a 2 mL well plate is used, the injection volume is 20 uL.

[0051] In this embodiment, the mobile phase of the liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A is configured as 0.1% formic acid water, and mobile phase B is configured as pure acetonitrile, and the separation is performed using a gradient elution procedure for 5 minutes at a flow rate of 0.4 mL / min. The specific elution gradients of the mobile phase A and the mobile phase B are as follows: Figure 3 , as shown in Table 1.

[0052] Table 1 Elution gradient

[0053] The mass spectrometry ESI source parameter configuration includes a capillary voltage of 1.0 kV, a desolvation gas temperature of 600 °C, and a desolvation gas flow rate of 1100 L / hr.

[0054] The MRM parameter configuration is shown in Table 2.

[0055] Table 2 MRM parameter data

[0056] S302. Preparation of calibration curve The calibration curve prepared was Figure 4 As shown, the calibration curve was used to quantify IGF-1 in the test samples.

[0057] First, prepare a high concentration mother solution. In this embodiment, preferably prepare a 20-fold high concentration mother solution 20*RL-H, then dilute the 20-fold high concentration mother solution successively to obtain 20-fold calibrators C1-C6, and finally dilute the 20-fold calibrators C1-C6 20 times to obtain calibrators C1-C6. The dilution concentration results are shown in Table 3.

[0058] Table 3 Concentration data of calibrators C1-C6

[0059] In this embodiment, a mother liquor diluent is used to dilute a 20-fold high concentration mother liquor. Preferably, the mother liquor diluent is 10% acetic acid, 30% methanol, and 60% water. First, 20*RL-H is diluted in sequence using the mother liquor diluent to obtain 20*C1, 20*C2, 20*C3, 20*C4, 20*C5, and 20*C6.

[0060] The preparation process is simpler by first preparing a high-concentration mother solution and then gradually diluting it into mother solutions of different concentrations according to a gradient. The multiple can be selected from 20 to 50 times, and 20 times is used as an example in this embodiment.

[0061] Then, 20*C1, 20*C2, 20*C3, 20*C4, 20*C5, and 20*C6 are diluted 20 times respectively using the first matrix diluent to obtain calibrators C1, C2, C3, C4, C5, and C6. Preferably, the first matrix diluent is 5% BSA + 0.1% Proclin300 preservative. The first matrix diluent is prepared by dissolving 5 g in 100 mL PBS and adding 100 uL Proclin300.

[0062] To ensure the consistency of the matrix, in this embodiment, a 20-fold high-concentration mother solution is first prepared, and then the high-concentration mother solution is diluted 20 times with the first matrix diluent to obtain a calibrator. In other embodiments, the calibrator can also be directly prepared.

[0063] The calibrators C1, C2, C3, C4, C5 and C6 were tested to obtain a calibration curve.

[0064] In this embodiment, a plurality of calibrators are used to prepare a calibration curve, so that the obtained standard curve is more stable and accurate for quantification.

[0065] S303. Detect the blood sample purified by SPE to obtain the content of IGF-1 The blood samples purified by SPE were tested by liquid chromatography-mass spectrometry, and the concentration of IGF-1 was quantified according to the calibration curve. Figure 5 shown.

[0066] Furthermore, in S302, it also includes preparing a linear reference and detecting the IGF-1 concentration in the linear reference based on the calibration curve.

[0067] Specifically, 20*C1, 20*C2, 20*C3, 20*C4, 20*C5, 20*C6, and 20*RL-H are diluted 20 times respectively with the second matrix diluent to obtain linear references RL1, RL2, RL3, RL4, RL5, RL6, and RL7. Preferably, the second matrix diluent is background-free human serum + 0.1% Proclin 300 preservative.

[0068] Each concentration of the linear reference was tested three times, and the results are shown in Table 4. The acceptable standard is that the correlation coefficient is ≥0.99 and the relative deviation (Bi) is ≤15%. As can be seen from Table 4, the coefficient of variation (CV) is less than 6% and the relative deviation is less than ±3%, indicating that the detection method of this embodiment has good linear performance.

[0069] Table 4 Linear reference test results

[0070] Furthermore, in S302, the IGF-1 concentration in SRM 2926 is detected based on the calibration curve.

[0071] Specifically, SRM2926 (NIST) was diluted 1000 times, the theoretical concentration was 305 ng / mL, and the test was performed three times. The results are shown in Table 5. The acceptable range is a relative deviation of ≤15%. As can be seen from Table 5, the relative deviation is 7.5%, which is much smaller than the maximum value of the acceptable range of ≤15%, indicating that the detection method of this embodiment has a high accuracy.

[0072] Table 5 SRM2926 test results

[0073] Furthermore, in S302, it also includes preparing a quantitative limit reference substance and detecting the IGF-1 concentration in the quantitative limit reference substance based on the calibration curve.

[0074] Specifically, 20*C1 was diluted 20 times with the second matrix diluent to obtain a quantitative limit reference, and the quantitative limit reference was detected 5 times, and the results are shown in Table 6. The acceptable range is a relative deviation of ≤15% and a coefficient of variation of ≤20%. As can be seen from Table 5, the CV of the quantitative limit reference is less than 5%, and Bi is less than 2%, indicating that the detection method of this embodiment has high sensitivity and can effectively detect IGF-1 components at a concentration of 10 ng / mL and above.

[0075] Table 6 Quantitation limit reference test results

[0076] The detection method of this embodiment only requires the collection and use of a trace amount of blood sample, meeting the quantitative limit of 10 ng / mL, which is far less than the requirement of 100uL usually required in the prior art.

[0077] Furthermore, to further verify the detection limit of this detection method, reference samples of 5 ng / mL, 2 ng / mL and 1 ng / mL were used for detection, and the results are shown in Table 7.

[0078] Table 7 Detection limit test results

[0079] Among them, %Bi and CV of 5ng / mL and 2ng / mL both meet the quantitative limit acceptance criteria (Bi≤15%, and CV≤20%); while %Bi>20% of 1ng / mL does not meet the quantitative limit requirements, which may be related to the fact that the signal-to-noise ratio is indeed not high enough and the baseline noise interference. Therefore, the quantitative limit of the detection method of this embodiment is determined to be 2ng / mL.

[0080] The insulin-like growth factor detection method based on QAMS collection in this embodiment is preferentially applicable to dried blood spot samples formed by peripheral blood. It only needs to collect a trace amount of blood sample and dry it to form a dried blood spot sample, and complete the drying and storage in an environment relatively independent of the outside world, thereby reducing the interference of the external environment on the sample and improving the stability of the sample, so that the detection result is closer to the actual IGF-1 concentration in the human body, especially when the blood sample is stored for a long time. In addition, the required detection limit is low and the sensitivity is high, so the collected trace sample can be effectively detected to ensure the accurate detection and quantification of IGF-1.

[0081] In addition to using dried blood spot samples as the basis for IGF-1 detection, the subsequent sample pretreatment and detection of insulin-like growth factor content using liquid chromatography-mass spectrometry in the present application can also be applied to liquid peripheral blood samples by appropriately adjusting the specific values ​​or conditions of the corresponding parameters.

[0082] Embodiment 2 As a specific embodiment of the present invention, this embodiment provides an insulin-like growth factor detection kit based on QAMS sampling, comprising: Internal standard working solution; Protein precipitants; as well as, Activating fluid; Balanced fluid; Eluent; eluent; The internal standard working solution is a diluent containing the N15 IGF-I internal standard, the protein precipitant is acetonitrile containing 5% acetic acid, the activation solution is methanol, the equilibrium solution is an aqueous solution containing 5% ammonia water, the eluent is an aqueous solution containing 5% ammonia water and / or 5% methanol water containing 1% acetic acid, and the eluent is a methanol: water: acetic acid mixed solution and / or pure water.

[0083] The kit is used for the detection of IGF-I.

[0084] The above description is only a preferred embodiment of the present invention and does not constitute any other form of limitation to the present invention. Any modification or equivalent change made based on the technical essence of the present invention still falls within the scope of protection required by the present invention.

Claims

1. A method for detecting insulin-like growth factor based on QAMS sampling, characterized in that: The method comprises: The peripheral blood samples of the sampling object are collected by using a quantitative absorption micro-sampler with integrated collection and storage to obtain 10uL-20uL equivalent dry blood spot samples; Pre-treating the dried blood spot sample, extracting a supernatant sample containing an insulin-like growth factor internal standard working solution, and releasing the insulin-like growth factor without using a surfactant, including transferring the dried blood spot sample to a centrifuge tube or a well plate and adding the prepared insulin-like growth factor internal standard working solution, adding an acetic acid-containing acetonitrile solution to remove protein after ultrasonication or oscillation, incubating at room temperature with vortexing and adding an ammonia solution to adjust the pH, obtaining a supernatant sample by centrifugation, and performing SPE purification on the extracted supernatant sample; Liquid chromatography-mass spectrometry is used to detect the insulin-like growth factor content of the supernatant sample extracted and SPE purified to obtain the insulin-like growth factor concentration, including configuring liquid chromatography-mass spectrometry, preparing a calibration curve and detecting the supernatant sample after SPE purification based on the calibration curve to obtain the content of insulin-like growth factor, wherein the mobile phase A and the mobile phase B of the chromatography are formic acid water and pure acetonitrile respectively, and gradient elution is used for separation.

2. The method for detecting insulin-like growth factor based on QAMS sampling according to claim 1, characterized in that: The internal standard working solution contains 0.1% BSA.

3. The method for detecting insulin-like growth factor based on QAMS sampling according to claim 1, characterized in that: The SPE purification includes activating and balancing the SPE column with methanol and ammonia solution, followed by secondary elution and secondary washing respectively; wherein the first elution uses 5% ammonia solution, the second elution uses 5% methanol solution containing 1% acetic acid, the first elution uses a methanol: water: acetic acid mixed solution, and the second elution uses pure water.

4. The method for detecting insulin-like growth factor based on QAMS sampling according to claim 1, characterized in that: The elution gradients of the mobile phase A and the mobile phase B are: Wherein, the mobile phase A is 0.1% formic acid water.

5. The method for detecting insulin-like growth factor based on QAMS sampling according to claim 1, characterized in that: The ESI source parameters of the mass spectrometer include a capillary voltage of 1.0 kV, a desolvation temperature of 600° C., and a desolvation flow rate of 1100 L / hr.

6. The method for detecting insulin-like growth factor based on QAMS sampling according to claim 1, characterized in that: The preparation of the calibration curve includes preparing a high-concentration mother solution, and diluting it with a mother solution diluent and a first matrix diluent to obtain a plurality of calibration products, and testing the plurality of calibration products to obtain a calibration curve, wherein the mother solution is 10% acetic acid, 30% methanol, and 60% water, and the first matrix diluent is 5% BSA+0.1% Proclin 300 preservative.

7. The method for detecting insulin-like growth factor based on QAMS sampling according to claim 1, characterized in that: The quantitative absorption micro-sampler contains a deoxidizing desiccant.

8. The method for detecting insulin-like growth factor based on QAMS sampling according to claim 1, characterized in that: The chromatographic column is Peptide C18 1.8um, 50mm; the injection volume is 10uL-20uL, and the flow rate of the mobile phase is 0.4mL / min.

9. An insulin-like growth factor detection kit based on QAMS sampling, characterized in that: It includes internal standard working solution, protein precipitant, activation solution, balancing solution, eluent and elution solution.

10. Use of the insulin-like growth factor detection kit based on QAMS sampling according to claim 9 in detecting insulin-like growth factor.

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